Compositions and methods for detecting pathogen infection

ABSTRACT

The present invention generally features therapeutic and diagnostic compositions and methods for increasing or decreasing the binding of a lysozyme polypeptide to a  Treponema pallidum  P17 polypeptide (Tp17) or a Tp17-like polypeptide. More particularly, the invention relates to compositions and methods for detecting, treating, or preventing a pathogen infection or a chronic disorder; and to binding assays using a Tp17-like polypeptide and a lysozyme polypeptide.

FIELD OF THE INVENTION

The present invention generally features diagnostic and therapeuticcompositions and methods derived from the characterization of thebinding of a lysozyme polypeptide to a Treponema pallidum P17polypeptide (Tp17) or Tp17-like polypeptides. In addition, the inventionprovides methods and compositions for increasing or decreasing thebinding between a lysozyme and a TP-17 or a TP-17 like polypeptide.

BACKGROUND OF THE INVENTION

Syphilis is a disease caused by Treponema pallidum (hereinafter alsoreferred to as “Tp”) infection. The diagnosis of syphilis is generallymade by an immunoassay of anti-Tp antibody in the blood using, forexample, the Treponema pallidum Hemagglutination Test (TPHA), theFluorescent Treponema Antibody Absorption Test (FTA-ABS) and/or theTreponema pallidum Immobilization Test (TPI), as well as enzyme-linkedimmuno sorbent assay (ELISA) and Western Blot systems. These testsdetect antibodies that react with Tp or antigen preparations from Tp,such as the Tp antigens Tp15, Tp17, and Tp47, for example. The TPI testinvolves a microscopic assessment of the extent to which complementactivating antibodies in a patient's serum inhibit the mobility of Tp.This test is not generally used as a diagnostic due to its high cost.The TPHA test involves agglutination of patient serum antibodies witherytirocytes to which the Tp sonicate antigen is bound. The FTA-ABS testinvolves indirect immunofluorescence microscopy to detect the binding ofspecific antibodies in patient serum to Tp attached to a solid supportvia a fluorescent-labelled secondary antibody. Although these assays arewidely used, they lack the sensitivity required for detecting earlystage or low level infection, when Tp antibody levels in the body fluidsare very low.

In addition, the popular use of individual recombinant or purified Tpantigens in existing immunoassays for syphilis does not account for thebinding by some antibodies to complexes formed between Tp antigens andother antigens (i.e., Tp antigen binding partners) normally present inthe subject's body fluid. Existing immunoassays thus fail to detect suchantibodies in the absence of the Tp antigen binding partner, whichresults in some instances in low sensitivity or false negative assayresults. A need therefore exists for pathogen diagnostic assays withimproved sensitivity, which detect antibodies that bind to pathogenantigen/binding partner complexes.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for the detection andtreatment of a pathogen infection. The invention generally relates tothe discovery that the Treponema pallidum polypeptide, Tp17, binds tolysozyme, an anti-microbial peptide produced by the host, and inhibitslysozyme's anti-microbial activity. The binding site on Tp17 forlysozyme and the binding site on lysozyme for Tp17 were identified.Comparisons of Tp17 sequences required for binding and inhibitinglysozyme with related proteins identified lysozyme binding consensussequences that are conserved among many different pathogens, including,for example, Herpes Simplex viruses, Hepatitis C virus, and parasitessuch as amoebas. This discovery suggests that pathogens share a generalmechanism for inhibiting a host immune response by producing apolypeptide containing a lysozyme binding motif that binds to lysozymeand inhibits its anti-microbial activity. Accordingly, the inventionalso provides for the identification of mutant lysozyme polypeptidescontaining mutations that interfere with or destabilize such binding.Such mutant lysozyme polypeptides, or fragments thereof, are useful inpreventing or treating a pathogen infection. In addition, the inventionprovides for improved diagnostic assays based on the detection ofantibodies that bind to a polypeptide-lysozyme complex, such as aTp17-lysozyme complex.

The identification of a lysozyme binding motif on Tp17, as well as theidentification of lysozyme binding motif consensus sequences shared byother pathogen-derived proteins, also provides useful compositions andmethods for the detection or inhibition of lysozyme, as well as thedetection of specific pathogens and inhibition of the correspondinginfections besides syphilis. In addition, pathogen-derived polypeptidesare useful in the diagnosis, prophylaxis, and treatment of a number ofdiseases, including diseases related to aberrant lysozyme activity.Those polypeptides also provide a useful affinity tag for use in proteinpurification using inexpensive lysozyme affinity chromatography.

In one aspect, the invention generally features a substantially purepolypeptide containing a lysozyme binding motif or a fragment thereof.In one embodiment, the invention provides a substantially pure Tp17-likepolypeptide, or a fragment thereof, containing at least a lysozymebinding motif containing an amino acid sequence of Xaa_(n) Pro HisXaa_(n) (SEQ ID NO:1), where Xaa is any amino acid and n is at least oneand the fragment is capable of binding a lysozyme polypeptide and wherethe motif is not CKPHDC (SEQ ID NO:24). In one embodiment, the fragmentis between about 4 amino acids and about 200 amino acids (e.g., 5, 10,25, 50, 75, 100, 125, 150, 175) of a Tp17-like polypeptide. In anotherembodiment, the fragment is less than about 30 or 100 amino acids of aTp17-like polypeptide. In another embodiment, the polypeptide or thefragment is capable of substantially inhibiting an enzymatic activity oran anti-microbial activity of the lysozyme. In yet another embodiment,the lysozyme binding motif contains an amino acid consensus sequenceCS1: Cys Xaa₁ Xaa₂ Xaa₃ Pro His Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys (SEQ ID NO:2), where Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅,Xaa₆, Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, or Xaa₁₃ is any amino acid,is absent, or is a peptide bond. Alternatively, the lysozyme bindingmotif contains an amino acid consensus sequence: Cys Xaa1 Xaa2 Arg Xaa3Xaa4 Xaa5 Cys (SEQ ID NO:314), where Xaa1, Xaa2, Xaa3, Xaa4, or Xaa5 isany amino acid, is absent, or is a peptide bond. Accordingly, a lysozymebinding motif of the present invention typically contains at least 4amino acids, 2 of which are selected from the group consisting of Cys,Pro, His, and Arg.

In related aspects, the invention features a substantially pure nucleicacid molecule encoding the polypeptide or the fragment of the previousaspect, a vector containing the nucleic acid molecule, and a host cellcontaining such a vector.

In another aspect, the invention features a substantially pure mutantlysozyme polypeptide containing at least one amino acid mutation thatreduces binding between the mutant lysozyme polypeptide and a Tp17-likepolypeptide (e.g., reduces binding by 5%, 10%, 25%, 50%, 75%, 85%, or95% relative to wild-type binding), where the mutant lysozymepolypeptide retains at least 50%, 60%, 75%, 85%, or 95% of theanti-microbial activity of a corresponding wild-type lysozymepolypeptide. In one embodiment, the mutation reduces binding between themutant lysozyme polypeptide and a Tp17-like polypeptide by at least 5%relative to wild-type lysozyme polypeptide binding. In anotherembodiment, the mutation affects a surface charge in the correspondingwild-type polypeptide. In other embodiments, the mutation is in apositively charged amino acid residue, is in a negatively charged aminoacid residue, or is in a hydrophobic amino acid residue of a wild-typelysozyme polypeptide that contacts a Tp17-like polypeptide. In otherembodiments, the mutation is of at least one, two, three, four, or moreamino acid positions selected from the group consisting of Lys19, Arg23,Lys51, Gly 55, Asn57, Arg131, Asn132 and Arg133 of human lysozyme or ispresent at a corresponding position in a lysozyme derived from anotherspecies (e.g., prokaryotic, eukaryotic, mammalian).

In a related aspect, the invention features a substantially pure nucleicacid molecule encoding the lysozyme polypeptide of the previous aspect,a vector containing such a nucleic acid molecule, and a host cellcontaining this vector.

In yet another aspect, the invention features a composition containing apolypeptide with at least a lysozyme binding motif and a lysozymepolypeptide. For example, a preferred composition contains asubstantially pure Tp17-like polypeptide and a substantially purelysozyme polypeptide, where the Tp17-like polypeptide is not the Ivypolypeptide. In one embodiment, the lysozyme is exogenous. In oneembodiment, the composition further contains carrier particles (e.g.,red blood cells, polypeptide aggregate particles, polymeric particles,inorganic particles, paramagnetic particles, and yeast cells).

In yet another aspect, the invention features a method for detecting animmune response against a pathogen in a subject. The method involves (a)contacting a biological sample from the subject with an exogenouslysozyme and a polypeptide containing a lysozyme binding motif (i.e., aTp17-like polypeptide); and (b) detecting antibody binding to thepolypeptide with a lysozyme binding motif or to a polypeptide-lysozymecomplex.

In yet another aspect, the invention features a method for enhancing thesensitivity of a diagnostic assay for detecting an immune responseagainst a pathogen. The method involves adding a lysozyme polypeptide tothe diagnostic assay (e.g., an agglutination assay), where the additionof the lysozyme polypeptide increases the assay's sensitivity by atleast 5%.

In various embodiments of the previous aspects, the lysozyme polypeptideis contacted with the Tp17-like polypeptide prior to, during, or aftercontacting the biological sample. In other embodiments, the assay is animmunoassay (e.g., enzyme-linked immunoabsorbent assay (ELISA), Westernblot, immunoagglutination assay, radioimmunoassay, turbidimetric assay,nephelometric assay, immunochromatographic assay, chemiluminescentassay, and fluorescent assay). In other embodiments, the biologicalsample is selected from the group consisting of blood, serum, plasma,tears, saliva, sputum, nasal fluid, ear fluid, genital fluid, breastfluid, milk, colostrum, placental fluid, perspiration, synovial fluid,ascites fluid, gastrointestinal fluid, exudate, transudate, pleuralfluid, pericardial fluid, amniotic fluid, cerebrospinal fluid, bile,gastric fluid, semen, fecal material, upper airway fluid, peritonealfluid, fluid harvested from a site of inflammation, fluid harvested froma pooled collection site, bronchial lavage, urine, biopsy material,aqueous humor, material from the forestomach of a ruminant animal,nucleated cell sample, any fluid associated with a mucosal surface,hair, and skin. In one preferred embodiment, the method is used todiagnose syphilis.

In another aspect, the invention features a composition for detecting apathogen in a sample, the composition containing a lysozyme polypeptideor a fragment thereof containing the amino acid sequence Xaa Xaa Xaa XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa Xaa XaaThr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly Xaa XaaGln Ile Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa CysAla Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa (SEQ IDNO:28), where Xaa is any amino acid or is absent, where the fragment iscapable of binding to a Tp17-like polypeptide.

In yet another aspect, the invention features a method for detecting oridentifying a pathogen or pathogen polypeptide. The method involves (a)contacting a sample with a lysozyme polypeptide fragment containing theamino acid sequence:       Xaa Xaa Xaa Xaa Xaa Xaa Cys (SEQ ID NO: 28)Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa Xaa Xaa Thr Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly Xaa Xaa Gln Ile AsnXaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys XaaXaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Ala Lys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp Xaa Xaa Xaa CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa,where Xaa is any amino acid or is absent, under conditions that permitbinding; and (2) detecting binding of the lysozyme polypeptide to apolypeptide in the sample, where the binding indicates the presence of apathogen or pathogen polypeptide in the sample. In another embodiment,the invention further involves determining the sequence of thepolypeptide bound to the lysozyme polypeptide. In some embodiments, thelysozyme polypeptide contains a sequence derived from a human lysozymepolypeptide.

In another aspect, the invention features a method for detectinglysozyme in a sample, the method involves (a) contacting the sample witha Tp17-like polypeptide bound to a solid support; and (b) detectingbinding of the polypeptide to a polypeptide in the sample, where thebinding indicates lysozyme is present in the sample (e.g., a biologicalfluid, a cell culture, a body sample, a water sample, a fluid sample, afood, a medicine, or a pathogen culture). In one embodiment, the methodfurther involves removing lysozyme from the sample through the binding.

In another aspect, the invention features a kit for detecting an immuneresponse to a pathogen containing a polypeptide having a lysozymebinding motif and a lysozyme polypeptide.

In another aspect, the invention features a kit for detecting a pathogenin a sample containing a labeled lysozyme polypeptide containing SEQ IDNO:28.

In another aspect, the invention features a fusion polypeptidecontaining at least a lysozyme binding motif (e.g., a fragment of aTp17-like polypeptide lysozyme binding motif) fused to a second aminoacid sequence with which it is not naturally linked. In one embodiment,the fusion polypeptide is fixed to a solid support. In anotherembodiment, the motif is at a terminus of the fusion polypeptide. Inanother embodiment, the fusion polypeptide is capable of reducing alysozyme anti-microbial activity.

In another aspect, the invention features a method for isolating afusion polypeptide, the method containing the steps of: (a) providingthe fusion polypeptide of the previous aspect; (b) contacting the fusionpolypeptide with a lysozyme polypeptide, where the lysozyme is affixedto a solid support, under conditions that permit binding of the fusionpolypeptide to the lysozyme polypeptide; and (c) eluting the fusionpolypeptide from the lysozyme polypeptide.

In another aspect, the invention features a method for identifying acandidate compound that increases lysozyme anti-microbial activity. Themethod involves detecting a reduction in binding between a lysozymepolypeptide and a polypeptide containing a lysozyme binding motif in thepresence of the candidate compound. In one embodiment, the candidatecompound reduces binding of a lysozyme binding motif to a catalyticglutamic acid that corresponds to Glu53 of human lysozyme.

In another aspect, the invention features a method for identifying amutant lysozyme polypeptide having increased anti-microbial activity.The method involves (a) providing a lysozyme polypeptide containing atleast one mutation in the amino acid sequence of the polypeptiderelative to a wild-type lysozyme sequence; and (b) detecting a reductionin binding between the mutant lysozyme polypeptide and a Tp17-likepolypeptides.

In yet another aspect, the invention features a method for treating orpreventing a pathogen infection in a subject in need thereof. The methodinvolves administering an effective amount of a mutant lysozymepolypeptide containing an amino acid mutation to a subject, where themutation reduces binding between the lysozyme polypeptide and aTp17-like polypeptide.

In another aspect, the invention features a method for treating orpreventing a pathogen infection in a subject in need thereof. The methodinvolves administering an effective amount of a mutant Tp17-likepolypeptide containing an amino acid mutation to the subject, where theadministration of the mutant Tp17-like polypeptide reduces the bindingbetween a pathogen-expressed Tp17-like polypeptide and an endogenouslysozyme polypeptide.

In another aspect, the invention features a vaccine that increases animmune response in a subject in need thereof, the composition containingat least one of the gJ and gD glycoproteins from Herpes Simplex-2 virus.

In another aspect, the invention features a vaccine that increases animmune response in a subject in need thereof, the composition containinga lysozyme binding polypeptide.

In another aspect, the invention features a vaccine that increases animmune response in a subject in need thereof, the composition containinga lysozyme polypeptide and a polypeptide containing a lysozyme bindingmotif.

In another aspect, the invention features a method of diagnosing aHerpes Simplex Virus type-2 infection in a subject. The method involvesdetecting the presence of a gJ protein lysozyme binding motif in asample from the subject. In one embodiment, the lysozyme binding motifis detected in a lysozyme binding assay. In another embodiment, thelysozyme binding motif is detected in an immunoassay.

In another aspect, the invention features a method for reducing lysozymeenzymatic activity in a sample. The method involves contacting a samplewith a Tp17-like polypeptide under conditions that permit binding of thepolypeptide to the lysozyme, where the Tp17-like polypeptide does notcontain the Ivy protein.

In another aspect, the invention features a method for reducing lysozymeenzymatic activity in a subject. The method involves administering tothe subject an effective amount of a Tp17-like polypeptide or fragmentthereof.

In various embodiments of the previous aspects, the polypeptide isHerpes Simplex Virus Type 2 glycoprotein J. In other embodiments of theprevious aspects, the method is used to treat or prevent a diseaseselected from the group consisting of cancer, Alzheimer's disease, renalamyloidosis, leukemia, Crohn's disease, and allergy.

In another aspect, the invention features a fragment of a substantiallypure Tp17-like polypeptide containing at least a lysozyme binding motifcontaining an amino acid sequence Cys Xaa₁ Xaa₂ Xaa₃ His Xaa₄ Xaa₅ Xaa₆Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys (SEQ ID NO:175), where Xaa isany amino acid or is absent and the fragment is capable of binding alysozyme polypeptide. In one embodiment, the fragment is derived fromPorphyromonas gingivalis or Helicobacter pylori.

In another aspect, the invention features a fragment of a substantiallypure APP-like polypeptide containing at least a lysozyme binding motifcontaining an amino acid sequence of Cys Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys (SEQ ID NO:177), where Xaa₁,Xaa₂, Xaa₃ Xaa₄ and Xaa₅ are any amino acid or are absent, Xaa₆ is aminoacid K, R or H, Xaa₇ is A or G, Xaa₈ is K, R, or H, Xaa₉ and Xaa₁₀ areany amino acid or are absent, Xaa₁₁ is amino acid K or R, Xaa₁₂ is anyamino acid or is absent, and Xaa₁₃ is amino acid E, D, Q or N. In oneembodiment, the APP-like polypeptide is the β-amyloid precursor protein.In another embodiment, the fragment is substantially a fragment of ahuman β-amyloid precursor protein.

In related aspects, the invention features nucleic acids encoding thefragments of the previous aspect, vectors containing the nucleic acids,and host cells containing such vectors.

In another aspect, the invention features a composition containing anAPP-like polypeptide and a substantially pure lysozyme polypeptide.

In another aspect, the invention features a method of diagnosingAlzheimer's disease in a subject. The method involves detecting thepresence in a biological sample (for example, a serum sample, acerebrospinal fluid sample, or a tissue sample) from the subject of acomplex between an APP-like polypeptide and a lysozyme polypeptide. Inone embodiment, method involves an immunoassay.

In another aspect, the invention features a method of diagnosing adisease, e.g., Alzheimer's disease by detecting the presence of anantibody that binds to a complex between an APP-like polypeptide and alysozyme polypeptide.

In another aspect, the invention features a method for identifying acandidate compound that modulates binding between the APP-likepolypeptide and a lysozyme polypeptide. The method involves detecting areduction in binding between the Tp17-like polypeptide and the lysozymepolypeptide in the presence of the candidate compound.

In another aspect, the invention features a Tp17-like polypeptidecontaining at least a lysozyme binding motif having an amino acidsequence of Xaa_(n) Pro His Xaa_(n) (SEQ ID NO:1), where Xaa is anyamino acid and n is at least one and the fragment is capable of bindingSLLP1. In one embodiment, the fragment is between about 4 amino acidsand about 200 amino acids of a Tp17-like polypeptide. In one embodiment,the motif contains the amino acid consensus sequence CS1: Cys Xaa₁ Xaa₂Xaa₃ Pro His Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys(SEQ ID NO:2), where Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈,Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, or Xaa₁₃ is any amino acid, is absent, or isa peptide bond.

In related aspects, the invention features a substantially pure nucleicacid molecule encoding the fragment of the previous aspect, a vectorcontaining the nucleic acid molecule, and a host cell containing thevector.

In another aspect, the invention features a substantially pure mutantSLLP1 polypeptide containing at least one amino acid mutation thatreduces binding between the mutant SLLP1 polypeptide and a Tp17-likepolypeptide relative to a wild-type SLLP1 polypeptide.

In another aspect, the invention features a substantially pure mutantSLLP1 polypeptide containing at least one amino acid mutation thatincreases the enzymatic activity of the mutant SLLP1 polypeptiderelative to a wild-type SLLP1 polypeptide. In one embodiment, theenzymatic activity includes an antimicrobial activity.

In another aspect, the invention features a method for modulatingfertility in a subject in need thereof. The method involvesadministering an effective amount of a mutant SLLP1 polypeptidecontaining at least one amino acid mutation to the subject, where theadministration of the mutant SLLP1 polypeptide alters the bindingbetween a Tp17-like polypeptide and an endogenous SLLP1 polypeptide suchthat the subject's fertility is modulated.

In another aspect, the invention features a method for modulatingfertility in a subject in need thereof. The method involvesadministering an effective amount of a Tp17-like polypeptide to thesubject, where the administration of the Tp17-like polypeptide reducesthe binding between an endogenous SLLP1 polypeptide and a cognatereceptor at an egg cell surface, such that the subject's fertility ismodulated.

In yet another aspect, the invention features a method for identifying acandidate compound that modulates human fertility. The method involvesdetecting a reduction in binding between a SLLP1 polypeptide and apolypeptide containing a lysozyme binding motif in the presence of thecandidate compound.

In another aspect, the invention features a method for treating orpreventing a sexually transmitted pathogen infection in a subject. Themethod involves administering an effective amount of a mutant SLLP1polypeptide containing at least one amino acid mutation to a subject,where the mutation reduces binding between an endogenous SLLP1polypeptide and a pathogen-expressed Tp17-like polypeptide.

In another aspect, the invention features a method for treating orpreventing a pathogen infection in a subject, e.g., a sexuallytransmitted pathogen infection. The method involves administering aneffective amount of a Tp17-like polypeptide to the subject, where theadministration of the Tp17-like polypeptide reduces the binding betweena pathogen-expressed Tp17-like polypeptide and an endogenous SLLP1polypeptide.

In another aspect, the invention features a method for identifying acandidate compound useful for the treatment or prevention of a pathogeninfection, e.g., a sexually transmitted pathogen infection. The methodinvolves (a) contacting a SLLP1 polypeptide and a Tp17-like polypeptidewith a candidate compound; and (b) detecting a reduction in bindingbetween the lysozyme polypeptide and the pathogen expressed Tp17-likepolypeptide in the presence of the candidate compound.

In another aspect, the invention features a method for reducing SLLP1binding to a sexually transmitted pathogen-expressed Tp17-like proteinin a subject. The method involves administering to the subject aneffective amount of a Tp17-like polypeptide or a lysozyme polypeptide,or a fragment thereof.

In another aspect, the invention features a contraceptive compositioncontaining a Tp17-like polypeptide that interferes with SLLP1 bindingduring a fertilization process.

In another aspect, the invention features a pharmaceutical compositionfor preventing or treating a sexually transmitted pathogen infection,the composition containing a Tp17-like polypeptide that interferes withSLLP1 binding during an infection process.

In another aspect, the invention features a pharmaceutical compositionfor use as an anti-microbial containing at least an effective amount ofbacteriophage T4 lysozyme or a lysozyme that is resistant to inhibitionby a Tp17-like polypeptide. In a preferred embodiment, thepharmaceutical composition further contains papain or bacitracin. In oneembodiment, pharmaceutical composition of the previous aspect, where thecomposition contains bacteriophage T4 lysozyme, papain, and bacitracin.In one embodiment, the amount of T4 or T4-like lysozyme present in aunit dose is between 2 mg and 100 mg (e.g., 5, 10, 20, 30, 40, 50, 60,70, 80, or 90). Preferably, the amount of T4 or T4-like lysozyme presentin a unit dose is 5 mg. In another embodiment, the amount of papainpresent in a unit dose is 2 mg. In one embodiment, the amount ofbacitracin present is 3 mg per unit dose. In another embodiment,composition is an oral formulation (e.g., a buccal tablet).

In another aspect, the invention features a method of treating apathogen infection in a subject involves administering to a subjectdiagnosed as having a pathogen infection a pharmaceutical compositioncontaining bacteriophage T4 lysozyme or a lysozyme that is resistant toinhibition by a Tp17-like polypeptide. In a preferred embodiment, thepharmaceutical composition further includes papain or bacitracin. In oneembodiment, the pharmaceutical composition contains bacteriophage T4lysozyme, papain, and bacitracin. In another embodiment, thepharmaceutical composition is in an oral formulation.

In various embodiments of any of the above aspects, the Tp17-likepolypeptide is derived from a pathogen, for example, a bacteria, avirus, a parasite, a plasmid, a prion, a mycoplasma, and a mycoticagent.

In other embodiments of any of the previous aspects, the pathogen is,for example, Escherichia coli, Pseudomonas aeruginosa, Pseudomonasputida, Yersinia pestis, Shigella flexnerii, Treponema denticola, Vibriocholerae, Vibrio vulnificus, Vibrio parahemolyticus, Chlamydiapneumoniae, Chlamydia trachomatis, Staphylococcus aureus, Staphylococcusepidermidis, Streptococcus pneumoniae, Streptococcus agalactiae,Streptococcus mutans, Streptococcus pyogenes, Enterococcus faecalis,Bordetella bronchiseptica, Bordetella pertussis, Bordetellaparapertussis, Helicobacter hepaticus, Helicobacter pylori, Salmonellatyphimurium, Ralstonia solanacearum, Xanthomonas campestris, Pseudomonassyringae, Pasteurella multocida, Brucella melitensis, Brucella suis,Porphyromonas gingivalis, Severe Acute Respiratory Syndrome (SARS)coronavirus, Respiratory Syncytial Virus, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Rubella virus, Toxoplasma gondii, Trypanozomaspp., Gardnerella vaginalis, Mycobacterium avium, Mycobacterium leprae,Mycobacterium paratuberculosis, Mycobacterium tuberculosis,Campylobacter jejuni, Helicobacter spp., Agrobacterium tumefaciens,Moraxella catarrhalis, Neisseria meningitidis, Neisseria gonorrhoeae,Haemophilus influenzae, Haemophilus ducreyii, Propionibacterium acnes,Listeria monocytogenes, Herpes Simplex Virus type 2, Influenza virus,TACARIBE virus, Bluetongue virus, Bacteroides thetaiotaomicron, Coxiellaburnetti, Legionella pneumophila, Salmonella typhi, Chimpanzeecytomegalovirus, Human cytomegalovirus, Human papilloma virus, Denguevirus, Foot and Mouth Disease, West Nile virus, Avian influenza virus,Human immunodeficiency virus, LdMNPV, Plasmodium falciparum, Plasmodiumovale, Emeiria tenella, Eimeria acervulina, Giardia lamblia, Plasmodiumyoelii and pathogens carried by Anopheles gambiae.

In other embodiments of any of the previous aspects, the pathogen is,for example, Treponema denticola, Bacteroides thetaiotaomicron, Coxiellaburnetti, Haemophilus influenzae, Neisseria gonorrhoeae, Legionellapneumophila, Staphylococcus, aureus, and Salmonella typhi, Neisseriameningitidis serogroup A and serogroup B, Vibrio cholerae, Vibriovulnificus, Haemophilus ducreyi, Hepatitis C virus, and Bordetellapertussis. Preferably, the pathogen is Treponema pallidum.

In other embodiments of any of the previous aspects, the motif containsthe amino acid consensus sequence CS2: Xaa1 Xaa2 Pro His Xaa3 Xaa4, (SEQID NO:3), where Xaa1 is Cys, Lys, Val, Ala, or is absent; Xaa₂ is Ala,Cys, or Lys; Xaa₃ is Ala, Asp, or Glu, and Xaa4 is Cys, Gly, or Lys. Inother embodiments of any of the previous aspects, the Tp17-likepolypeptide contains the sequence Cys Xaa₁ Xaa₂ Xaa₃ Pro His Xaa₄ Xaa₅Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys (SEQ ID NO:2), whereXaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁,Xaa₁₂, or Xaa₁₃ is any amino acid or is absent. In still otherembodiments of any of the previous aspects, the motif contains an aminoacid consensus sequence CS3: Xaa Cys Pro His Ala Gly (SEQ ID NO:25),where Xaa is Cys or Val. In yet other embodiments of any of the previousaspects, the motif is selected from the group consisting of CCPHAG (SEQID NO:4), VCPHAG (SEQ ID NO:5), VAPHDC (SEQ ID NO:6), KAPHDK (SEQ IDNO:7), VKPHDG (SEQ ID NO:8); KKPHAK (SEQ ID NO:9), KAPHEK (SEQ IDNO:10), KKPHAC (SEQ ID NO:11), VAPHAG (SEQ ID NO:12), VKPHAK (SEQ IDNO:13), VKPHAC (SEQ ID NO:14), VAPHEG (SEQ ID NO:15), VKPHEK (SEQ IDNO:16), VCPHEK (SEQ ID NO:17), CKPHAG (SEQ ID NO:18), ACPHAG (SEQ IDNO:19) or KCPHDC (SEQ ID NO:20), VKPHDK (SEQ ID NO:21), KKPHAG (SEQ IDNO:22), and CAPHEK (SEQ ID NO:23).

In alternative embodiments of any of the previous aspects, the lysozymebinding motif contains an amino acid sequence of Cys Xaa1 Xaa2 Arg Xaa3Xaa4 Xaa5 Cys (SEQ ID NO:314), where Xaa1 is Pro, Ala, Val, or Ser; Xaa2is Asp, Glu, or His; Xaa3 is Leu or Met; Xaa4 is Ser, Ala, or Gly; andXaa5 is Ser, Val, Ala, Lys, or Cys. In other embodiments, the lysozymebinding motif contains an amino acid sequence of Cys Pro Xaa1 Arg Xaa2Xaa3 Xaa4 Cys (SEQ ID NO:315), where Xaa1 is Asp, Glu, or His; Xaa2 isLeu or Met; Xaa3 is Ser or Ala; Xaa4 is Ser, Ala, or Val. In yet otherembodiments, the lysozyme binding motif contains an amino acid sequenceselected from the group consisting of CPHRLSVC (SEQ ID NO:316), CPHRLSSC(SEQ ID NO:317), CPERLASC (SEQ ID NO:318), CPERMASC (SEQ ID NO:319),CPERLSSC (SEQ ID NO:320), and CPQRLSSC (SEQ ID NO:321). One exemplarylysozyme binding motif contains an amino acid sequence of CPHRLSVC (SEQID NO:316).

In various embodiments of any of the above aspects, the lysozymepolypeptide contains the following amino acid sequence: Xaa Xaa Xaa XaaXaa Xaa Cys Xaa (SEQ ID NO: 28) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa GluSer Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa AspTyr Gly Xaa Xaa Gln Ile Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa TrpXaa Xaa Trp Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys XaaXaa Xaa,where Xaa is any amino acid or is absent.

In other embodiments of any of the previous aspects, the polypeptidecontaining a lysozyme binding motif (i.e., a Tp17-like polypeptide) orthe lysozyme polypeptide, or a fragment thereof, is isolated from abiological sample, is a recombinant or chemically synthesizedpolypeptide, is fused to an affinity tag, is affixed to a solid support(e.g., a resin, a gel, a bead, a well, a column, a chip, a membrane, amatrix, a plate, and a filter device), is derived from a humanpolypeptide, is detectably labeled (e.g., labeled with a fluorophore, afluorescent protein, a chromophore, a radioactive moiety, a luminiferousmoiety, and an enzymatically active label). In still other embodiments,the lysozyme polypeptide and the Tp17-like polypeptide are present in amolecular ratio of between about 0.001 and about 1,000,000.

Compositions and methods of the invention are useful for identifyingand/or quantifying antibodies to pathogens such as, for example,Treponema pallidum, Escherichia coli (e.g., E. coli O157:H7 and E. coliK1), Pseudomonas aeruginosa, Pseudomonas putida, Yersinia pestis,Shigella flexnerii, Treponema denticola, Vibrio cholerae, Vibriovulnificus, Vibrio parahemolyticus, Chlamydia pneumoniae, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus pneumoniae,Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Enterococcus faecalis, Bordetella bronchiseptica, Bordetella pertussis,Bordetella parapertussis, Helicobacter hepaticus, Salmonellatyphimurium, Ralstonia solanacearum, Xanthomonas campestris, Pseudomonassyringae, Pasteurella multocida, Brucella melitensis, Brucella suis,Mycobacterium tuberculosis, Campylobacter jejuni, Helicobacter spp.,Agrobacterium tumefaciens, Moraxella catarrhalis, Neisseriameningitidis, Neisseria gonorrhoeae, Mycoplasma pneumoniae, Legionellasp., Haemophilus influenzae, Haemophilus ducreyii, Propionobacteriumacnes, Listeria monocytogenes, Herpes Simplex Virus type 2, Influenzavirus, TACARIBE virus, Hepatitis A virus, Hepatitis B virus, Hepatitis Cvirus, Bluetongue virus, Chimpanzee cytomegalovirus, Humancytomegalovirus, Human papilloma virus, Dengue virus, Foot and MouthDisease, Human immunodeficiency virus, cattle plague virus, rabiesvirus, LdMNPV, Entamoeba histolytica, Plasmodium falciparum, Plasmodiumovale, Emeiria tenella, Eimeria acervulina, Giardia lamblia, Plasmodiumyoelii, and pathogens carried by Anopheles gambiae. In anotherembodiment, pathogens such as Mycobacterium paratuberculosis, Ebolavirus, Rift Valley Fever virus, Severe Acute Respiratory Syndrome (SARS)virus, Small Pox virus, Bacillus anthracis, Leishmania Spp., mycoplasma,rickettsia, fungi, and yeast are detected.

In another embodiment, the invention provides compositions for detectinganti-pathogen antibodies in a sample, comprising (a) a polypeptidecomprising the amino acid sequence of TAPHRGLATLYNGDC (SEQ ID NO:26) orCSPEVGQMDC (SEQ ID NO:27), and (b) a full-length lysozyme polypeptide ora fragment thereof.

In an embodiment, lysozyme (A) and Tp17-like polypeptides (B) arepresent in a molar ratio (A/B) of between about 0.001 and about1,000,000. In a preferred embodiment, lysozyme (A) and Tp17-likepolypeptides (B) are present in a molar ratio (A/B) of between about0.01 and about 1,000,000. In another preferred embodiment, lysozyme (A)and Tp17-like polypeptides (B) are present in a molar ratio (A/B) ofbetween about 0.1 and about 1,000,000. Most preferably, lysozyme (A) andTp17-like polypeptides (B) are present in a molar ratio (A/B) of betweenabout 1 and about 1000, between about 10 and about 1000, or betweenabout 100 and about 1000.

The methods of the invention are used to diagnose diseases such as, forexample, syphilis, HIV infection, genital herpes, bubonic plague,rabies, amoebic, dysentery, shigellosis, dental caries, E. coliinfection, cystic fibrosis, tuberculosis, listeriosis, cholera, group Aor group B streptococcal infections, staphylococcal infections, gastriculcer, whooping cough, Enterococcal infections, chlamydiosis,brucellosis, whooping cough, otitis media, otitis interna, meningitis,mycoplasmosis, mycosis, influenza infection, malaria, Hepatitis B, andHepatitis C virus infections. In another embodiment, the methods of theinvention are used to diagnose salmonellosis, gonorrhea, vibriosis,colibacillosis, pneumonia, typhus, typhoid fever, amoebiasis,bronchitis, severe acute respiratory syndrome (SARS), Legionaire'sdisease, and cytoplasmic storage diseases, such as lysosomal storagediseases.

The invention also provides methods for detecting lysozyme in a sampleby contacting the sample with a Tp17-like polypeptide and detecting thebinding of the Tp17-like polypeptide to the lysozyme. Lysozymeanti-microbial activity is reduced by contacting a sample with aTp17-like polypeptide under conditions that permit binding of theTp17-like polypeptide to the lysozyme. The sample may be a pathogenpreparation, such as, for example, a bacteria, a virus, a parasite, aplasmid, a mycoplasma, a mycotic agent (e.g., fungus and yeast), and aprion preparation. The sample may also be a material that needs to berendered free of lysozyme contamination, such as a biological sample(e.g., throat aspirate, hemoculture, cerebrospinal fluid), culturevessel, cell culture, cuvette, swab, clinical diagnostic apparatus orassay material, medical instrument, fluid, water, food, medicine,implant, or graft.

The methods of the invention may also, be used for reducing lysozymeanti-microbial activity in a subject (e.g., an animal, such as human) byadministering to the subject an effective amount of a Tp17-likepolypeptide. The methods are used to treat or prevent diseases such ascancer, infectious disease, inflammatory disease, Alzheimer's disease,renal amyloidosis, leukemia, Crohn's disease, and allergy, which areassociated with increased lysozyme activity over normal levels.

In another aspect, the invention provides compositions for detecting apathogen in a sample, comprising a ligand, or a binding partner, capableof binding to a polypeptide with a lysozyme binding motif (i.e., aTp17-like polypeptide). In an embodiment, the ligand comprises alysozyme polypeptide, a monoclonal antibody, a polyclonal antibody, oran Fab fragment, or DNA or RNA aptamers, for example. In an embodiment,the ligand comprises a detection molecule.

In another aspect, the invention provides methods for detecting apathogen in a sample by (1) contacting a sample with a ligand (e.g., alysozyme polypeptide) capable of binding to a polypeptide with alysozyme binding motif (i.e., a Tp17-like polypeptide) under conditionsthat permit binding of the ligand to a pathogen or a pathogenpolypeptide; and (2) detecting the binding of the ligand to the pathogenor pathogen polypeptide, if present, wherein such binding is indicativeof the presence of the pathogen or pathogen polypeptide in the sample.In an embodiment, the ligand is labeled with a detection molecule, suchas a fluorophore, a fluorescent protein, chromophore, radioactivemoiety, luminiferous moiety or enzymatically active reporter.

In another aspect, the invention provides a kit comprising a polypeptidewith a lysozyme binding motif (i.e., a Tp17-like polypeptide) and aligand capable of binding to the polypeptide with a lysozyme bindingmotif. In an embodiment, the ligand is attached to a solid support suchas a resin, bead, well, chip, column, gel, membrane, or filter device.In a preferred embodiment, the ligand is a lysozyme polypeptide.

In another aspect, the invention provides recombinant fusion proteinscomprising a polypeptide with a lysozyme binding motif (i.e., aTp17-like polypeptide) linked to a polypeptide of interest. In anembodiment, the Tp17-like polypeptide is at the N or C terminus of thepolypeptide of interest. In another embodiment, the Tp17-likepolypeptide is between the N or C terminus of the polypeptide ofinterest. In an embodiment, the recombinant protein is capable ofreducing lysozyme activity. The invention further provides methods ofpreparing the recombinant fusion proteins by linking the Tp17-likepolypeptide to the polypeptide of interest. Yet further, the inventionprovides methods of using the recombinant fusion proteins to affinitypurify a polypeptide of interest by (1) contacting a sample comprising arecombinant fusion polypeptide with lysozyme under conditions thatpermit the polypeptide to bind to lysozyme; (2) washing the sample; and(4) eluting the recombinant fusion polypeptide from the lysozyme. In anembodiment, the method comprises isolating the polypeptide of interestfrom the Tp17-like polypeptide. In an embodiment, the lysozyme is linkedto a solid support.

In another aspect, the invention provides methods for treating orpreventing a disease associated with a pathogen infection byadministering to a subject in need thereof an effective amount of alysozyme that is resistant to inhibition by a Tp17-like polypeptide. Thelysozyme that is resistant to inhibition by a Tp17-like polypeptide canbe selected from naturally-occurring lysozymes and modified lysozymes.In some embodiments, the method for treating or preventing a pathogeninfection in a subject comprising administering an effective amount of alysozyme comprising an amino acid sequence selected from the groupconsisting of Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly XaaXaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Gly (SEQ ID NO:322); and Asp Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn (SEQ ID NO:323),where Xaa is any amino acid or is absent. In other embodiments, themethod for treating or preventing a pathogen infection in a subjectcomprising administering an effective amount of a lysozyme comprising anamino acid sequence selected from the group consisting of Tyr Tyr LeuThr Xaa Phe Cys Xaa Xaa Xaa Xaa Ala Cys Gly Xaa Xaa Xaa Xaa Cys Ser XaaXaa Xaa Tyr Phe Thr Ala Asp Ser Gln Arg Phe Gly Cys Gly (SEQ ID NO:324);and Asp Ala Gly Pro Xaa Xaa Xaa Val Glu Xaa Xaa Ala Gly Xaa Xaa Ile IleAsp Ala Ser Xaa Xaa Ile Cys Xaa Xaa Leu Xaa Gly Xaa Ser Ser Cys Gly TrpSer Asp Xaa Xaa Xaa Ile Thr Ala Xaa Xaa Xaa Ser Xaa Xaa Asp Xaa Xaa ProXaa Xaa Xaa Pro Phe Asn Val Thr (SEQ ID NO:325), where Xaa is any aminoacid or is absent. The present invention also relates to apharmaceutical composition or a kit for use as an anti-microbialcontaining at least an effective amount of a lysozyme with anabove-described amino acid sequence.

In another aspect, the invention provides methods for treating orpreventing a disease associated with a pathogen infection byadministering to a subject in need thereof an effective amount of anreagent that inhibits the binding of a polypeptide with a lysozymebinding motif (i.e., a Tp17-like polypeptide) to a lysozyme polypeptide.In one embodiment, the reagent inhibits binding of a Tp17-likepolypeptide to the glutamic acid 53 of human or chicken lysozyme or to acorresponding amino acid residue in other species (numbering refers tothe full-length human lysozyme sequence deposited under accession numberNP_(—)000230 (SEQ ID NO:302)). In another embodiment, the reagent is apeptide, chemical drug, antibody, nucleic acid, PNA, small interferingRNA, or bacteriophage, DNA aptamer, or RNA aptamer. In anotherembodiment, the reagent is a lysozyme ligand.

The invention also provides treatments or vaccines comprising at leastone of the gJ and gD glycoproteins from Herpes Simplex-2 virus. Theinvention also provides vaccines comprising (a) a lysozyme ligand and/or(b) a lysozyme polypeptide.

By “Tp17-like polypeptide” is meant an amino acid sequence, or fragmentthereof, that comprises a lysozyme binding motif that has the ability tobind to a lysozyme polypeptide and may inhibit a biological activity oflysozyme. In one embodiment, the lysozyme binding motif comprises aXaa_(n) Pro His Xaa_(n) (SEQ ID NO:1) consensus sequence. In particular,a Tp17-like polypeptide comprises a Xaa_(n) Pro His Xaan (SEQ ID NO:1)consensus sequence and is at least 50%, 75%, 85%, 95%, or 99% identicalto Tp17 (Genbank Accession No. P29722 (SEQ ID NO:31)) or a fragmentthereof. In other embodiments, the lysozyme binding motif comprises thefollowing consensus sequence:CX(1,5)[KRH][AG][KRH]X(0,2)[KR]X(0,1)[EDQN]C (SEQ ID NO:178). In yetother embodiments, the lysozyme binding motif comprises the followingconsensus sequence: Cys Xaa1 Xaa2 Arg Xaa3 Xaa4 Xaa5 Cys (SEQ IDNO:314). Tp17-like polypeptides include, but are not limited to, allvariants, homologs, and mutants of Tp17 or other naturally-occurringproteins containing a lysozyme binding motif, full length or fragmentsthereof, and fusion proteins containing any of the above as a materialpart. Exemplary Tp17-like polypeptides are provided in FIGS. 1, 2, 39,40, and 42.

By “mutant Tp17-like polypeptide” is meant a TP-17-like polypeptidehaving at least one amino acid change relative to the sequence of anaturally-occurring sequence. Such changes include, for example, aminoacid substitutions, deletions, or insertions. In some embodiments,mutant Tp17-like polypeptide homologs, mutants, fragments, substitutionsand modifications retain the ability to bind to lysozyme. In onepreferred embodiment, such polypeptides bind lysozyme, but fail toinhibit its anti-microbial activity. FIG. 7 describes exemplary methodsfor targeted and random identification of polypeptide mutants.

By “Tp17-like nucleic acid molecule” or “mutant Tp17-like nucleic acidmolecule” is meant a nucleic acid sequence encoding a Tp17-like ormutant Tp17-like polypeptide, respectively.

By “APP-like polypeptide” is meant a polypeptide that binds lysozyme andthat contains the following consensus sequence:

-   -   CX(1,5)[KRH][AG][KRH]X(0,2)[KR]X(0,1)[EDQN]C (SEQ ID No:178).

By “lysozyme” or “lysozyme polypeptide” is meant a polypeptide havinglysozyme activity including anti-microbial activity or enzymaticactivities. In one embodiment, lysozyme or lysozyme polypeptidecomprises the following consensus sequence       Xaa Xaa Xaa Xaa Xaa XaaCys (SEQ ID NO: 28) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser XaaXaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr GlyXaa Xaa Gln Ile Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Cys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa TrpXaa Xaa Trp Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Xaa Xaa Xaa.

Exemplary lysozyme polypeptides are listed in FIGS. 3A and 3B. In onepreferred embodiment, a lysozyme polypeptide is at least 50%, 75%, 85%,95%, or 99% identical to human lysozyme (Genbank Accession No.NP_(—)000230 (SEQ ID NO:302), NP_(—)783862 (SEQ ID NO:303), NP_(—)115906(SEQ ID NO:304), NP_(—)898881 (SEQ ID NO:305), NP_(—)653235 (SEQ IDNO:306), NP_(—)065159 (SEQ ID NO:307), NP_(—)776246 (SEQ ID NO:308),NP_(—)995328 (SEQ ID NO:309), NP_(—)002280 (SEQ ID NO:310)). In otherembodiments, lysozyme or lysozyme polypeptide comprises at least one ofthe following consensus sequences: Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa (SEQID NO: 322) Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly; or Asp Ala Xaa Xaa Xaa XaaXaa Xaa (SEQ ID NO: 323) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa XaaXaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa XaaXaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Asn,where Xaa is any amino acid or is absent.

By “mutant lysozyme polypeptide” is meant a lysozyme amino acid sequencecontaining at least one amino acid change relative to a naturallyoccurring lysozyme amino acid sequence.

By “lysozyme nucleic acid molecule” or “mutant lysozyme nucleic acidmolecule” is meant a nucleic acid molecule encoding a lysozyme or mutantlysozyme polypeptide, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention, as well as the invention itself, will be more fullyunderstood from the following description of preferred embodiments whenread together with the accompanying drawings, in which:

FIGS. 1A and 1B illustrate the lysozyme binding sites on Tp17-likepolypeptides from a number of bacteria, viruses, parasites and mammalianorganisms and the 17 amino acid consensus sequence they define. In FIG.1B, Tp17-related polypeptides from T. denticola, B. thetaiotaomicron, C.burnetti, H. influenzae, N. meningitidis serogroup A and serogroup B, V.cholerae, V. vulnificus, H. ducreyi, S. typhi, L. pneumophila, S.aureus, N. gonorrhoeae, and B. pertussis fit the following consensussequence: Cys Xaa1 Xaa2 Xaa3 Pro His Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10Xaa11 Xaa12 Xaa13 Xaa14 Xaa15 Xaa16 Xaa17 Cys (SEQ ID NO:176) whereinXaa is any aminoacid or is absent. The two remaining proteins from P.gingivalis, and H. pylori are grouped under the following consensus:sequence Cys Xaa1 Xaa2 Xaa3 His Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10Xaa11 Xaa12 Xaa13 Cys (SEQ ID NO: 175), wherein x is any amino acid oris absent. FIG. 1B is a table defining a lysozyme binding motifconserved among a variety of clinically relevant bacterial pathogens.

FIG. 2 illustrates the lysozyme binding sites on Tp17-like polypeptidesfrom a number of bacteria, viruses, parasites, insects and mammalianorganisms and the 6 amino acid consensus sequence they define.

FIG. 3A is an alignment of mature lysozyme sequences from a number ofspecies. E35 and D53 in mature chicken lysozyme (or D₅₂ in mature humanlysozyme) (▾) belong to the catalytic dyad and are conserved across allknown lysozyme sequences. A consensus sequence is shown at the bottom ofthe alignment. Amino acid conservation is represented according toRisler comparison symbols (Risler et al. (1988) J. Mol. Biol. 204:1019).In that representation system, X represents any amino acid or no aminoacid, upper case letters correspond to 100% amino acid conservation,lower-case letters correspond to a value of amino acid conservationcomprised between 50% and 90%. Moreover, conservative changes shared bymore than 50% of the sequences are represented using the followingsymbols $ corresponds to leucine or methionine, ! corresponds toisoleucine or valine, and # corresponds to asparagine or aspartic acid.

FIG. 3B is a continuation of the alignment of mature lysozyme sequencespresented in FIG. 3A. A consensus sequence is shown at the bottom of thealignment using the nomenclature described with respect to FIG. 3A.

FIG. 4 illustrates an advantage of the present invention sinceconventional Tp17 antibody detection kits do not detect antibodies tothe Tp17-lysozyme complex. In one embodiment, the invention features akit containing a Tp17-like polypeptide and a lysozyme polypeptide. Suchkits are capable of detecting antibodies to Tp17 alone, lysozyme alone,or a Tp17-lysozyme complex, if present.

FIG. 5 illustrates a far western blot lysozyme-protein interaction assaycomprising the following five steps: (1) immobilization of purifiedlysozyme onto a solid support; (2) probing of the solid support with aligand for lysozyme (e.g., GST-Tp17); (3) binding of GST-Tp17 with goatanti-GST antibody; (4) binding of the goat antibody with an anti-goatalkaline phosphatase conjugate; and (5) staining with nitrobluetetrazolium/bromochloro indolyl phosphate alkaline phosphatasechromogenic substrate (BT/BCIP).

FIG. 6 illustrates the amino acid (SEQ ID NO:29) and nucleotide (SEQ IDNO:30) sequence of GST-Tp17 fusion protein. The sequences correspondingto the GST moiety and to Tp17 are represented in italics and bold,respectively. Numbered lines, SEQ ID NO: 30, represent the sense DNAstrand; the antisense or RNA strand is below the sense strand.

FIG. 7 illustrates the Tp17-HIS expression construct pET24a_Tpp17His.This vector derives from the pET24 expression vector (Novagen Inc.,Madison, Wis.).

FIG. 8A is a Coomasie Brilliant Blue-stained gel of purified GST-Tp17chromatographic fractions. Lane 1: purification process with addedchicken lysozyme; lane 2: purification process without added chickenlysozyme; lane 3: MWM: molecular weight markers.

FIG. 8B is a Western Blot and Coomasie Brilliant Blue-stained gel ofpurified Tp17-HIS chromatographic fractions. Lane 1: Western blot usinga syphilis positive human serum of proteins recovered from thepurification process in the presence of added chicken lysozyme; lane 2:Coomasie Brilliant Blue staining of proteins recovered from thepurification process in the presence of added chicken lysozyme. Thepeptide sequences obtained by N-terminal amino acid sequencing of theproteins recovered from the electrophoretic bands in lanes one and twoare indicated.

FIG. 9A illustrates the results from probing chicken lysozyme withGST-Tp47 in a far western blot assay. Lane MW: molecular weight markers;lane 1: 1 μg purified chicken lysozyme; lane 2: 5 μg purified chickenlysozyme; lane 3: 10 μg purified chicken lysozyme.

FIG. 9B illustrates the results from probing chicken lysozyme withGST-Tp17 in a far western blot assay. Lane MW: molecular weight markers;lane 4: 1 μg purified chicken lysozyme; lane 5: 5 μg purified chickenlysozyme; lane 6: 10 μg purified chicken lysozyme.

FIG. 9C illustrates the results from probing human lysozyme withGST-Tp47 in a far western blot assay. Lane MW: molecular weight markers;lane 7: 1 μg purified human lysozyme; lane 8: 5 μg purified humanlysozyme; lane 9: 10 μg purified human lysozyme.

FIG. 9D illustrates the results from probing human lysozyme withGST-Tp17 in a far western blot assay. Lane MW: molecular weight markers;lane 10: 1 μg purified human lysozyme; lane 11: 5 μg purified humanlysozyme; lane 12: 10 μg purified human lysozyme.

FIG. 10 shows a graphical representation of the effect of Tp17polypeptide on lysozyme antibacterial activity. The concentration ofGST-Tp17 in the presence of human (O) and chicken (▴) lysozyme wasplotted versus relative fluorescence intensity. As a control, theconcentration of GST-Tp47 in the presence of human (♦) lysozyme was alsoplotted versus relative fluorescence intensity.

FIG. 11A shows an alignment of the amino acid sequence of the IVYprotein from E. coli with the amino acid sequence of Tp17 from T.pallidum. A consensus sequence is shown under the alignment. “#”identifies conservatives (Asp/Glu) and semi-conservatives (Asp/Asn)changes whereas “!” indicates Val/Ile changes.

FIG. 11B illustrates the crystal structure of chicken lysozyme's threepossible contact regions with E. coli IVY:protein: peptides I, II, andIII are indicated with dark shading.

FIG. 12 illustrates atomic interactions between E. coli Ivy and chickenlysozyme, including the interaction between the nitrogen atom of H88-ivyand the oxygen atom of E53-chkcLys and between the oxygen atom ofD89-ivy and the nitrogen atom of R132-chkcLy. The distance betweenatomic centers was 2.56 Å (1 angstrom=10-10 meter) in theH88-ivy/E53-chkcLys interaction and 2.46 Å in the D88-ivy/R132-chkcLysinteraction.

FIG. 13A illustrates the water accessible, molecular surface (light) ofchicken lysozyme alone. The surface-exposed, water accessible region ofamino acid residue E53 is shaded.

FIG. 13B illustrates the water accessible molecular surface of chickenlysozyme complexed to E. coli IVY. Amino acid residue E53 is completelymasked and poorly accessible to the solvent.

FIG. 13C illustrates the water accessible, molecular surface (light) ofchicken lysozyme. The surface-exposed, water accessible region of aminoacid residue R132 is shaded.

FIG. 13D illustrates the water accessible molecular surface of chickenlysozyme complexed to E. coli VY. Amino acid residue R132 remainspartially exposed to the solvent

FIG. 14A illustrates the bridging of the two flanking cysteines of theE. coli IVY-chicken lysozyme binding site that brings the histidine andglutamic acid residues in close spatial proximity, in a configurationable to make contact with residues E53 and R132 of chicken lysozyme.

FIG. 14B illustrates the bridging of the two flanking cysteines of theT. pallidum Tp17-lysozyme binding site that likely brings the histidineand aspartic acid residues in close spatial proximity, possibly in aconfiguration able to make contact with residues E₅₃ and R₁₃₂ oflysozyme.

FIG. 15 illustrates an alignment of protein gJ from Herpes simplex virustype 1 (HSV1) and Herpes simplex virus type 2 (HSV2). A consensussequence region is magnified under the alignment. The insertdemonstrates that HSV1 gJ does not share the Tp17-like consensussequence found in HSV2 gJ. “#” identifies semi-conservatives changessuch as Asp/Asn.

FIG. 16A is a table illustrating the relative hemagglutination intensitycorresponding to 4 borderline human syphilitic sera (HPM10, HG38, HG48,and HG82). Serum samples were combined with Tp17 (R) in the presence orabsence of chicken lysozyme (ChickenLyz) or human lysozyme (HumanLyz).Results are expressed as reciprocal dilution titers (e.g., 80 meanspositive at dilution 1/80).

FIG. 16B is a graphical representation of the hemagglutination resultsof FIG. 16A. Tp17 reagent without lysozyme (black bars); Tp17 reagentsupplemented with chicken lysozyme (hatched bars); Tp17 reagentsupplemented with human lysozyme (empty bars). Results are expressed asreciprocal dilution titers (e.g., 80 means positive at dilution 1/80).

FIG. 17A is a table illustrating the relative hemagglutination intensitycorresponding to 10 human syphilitic sera (Syph71, 09-4, 09-3, HSP91,HSP17, HSP3, 4932, 5895, HG62, and HG82). Serum samples were combinedwith Tp17 (R) in the presence or absence of natural human lysozyme(LyzN) or recombinant human lysozyme (LyzR). Results are expressed asreciprocal dilution titers (e.g., 80 means positive at dilution 1/80).

FIG. 17B is a graphical representation of the hemagglutination resultsof FIG. 17A. Tp17 reagent without lysozyme (black bars); Tp17 reagentsupplemented with natural human lysozyme (checked bars); Tp17 reagentsupplemented with recombinant human lysozyme (empty bars). Results areexpressed as reciprocal dilution titers (e.g., 80 means positive atdilution 1/80).

FIG. 18A illustrates the improvement of the relative intensity ofhemagglutination with the addition of recombinant human lysozyme. Atotal of 34 sera were tested using either a Tp17 reagent (R) or Tp17reagent supplemented with recombinant human lysozyme (R+LyzR). Theresults of the technique are given as the inverse of the last dilutionthat gave a positive agglutination. Each result was converted into anumber (1/80 dilution is 1, 1/160 dilution is 2, etc.). The cut-off isat dilution 1/80 and a value lower than 1 is considered a negativeresult. FN and P respectively stand for False-negative and positiveresults, respectively.

FIG. 18B illustrates the relative intensity of hemagglutination with theaddition of recombinant human lysozyme on false negative serum samplesusing either Tp17 reagent (R) or Tp17 reagent supplemented withrecombinant human lysozyme (R+LyzR). The intensity of the agglutinationin the false negative samples is represented by a number between 0 and 1(1 represents the cut-off of the hemagglutination technique).

FIG. 19 illustrates an hemagglutination assay performed with 34 humansera. The assay was performed using a Tp17 reagent (black bars) or aTp17 reagent supplemented with recombinant human lysozyme (empty bars).The result of the technique is given by the inverse of the last dilutionthat gave a positive agglutination. The cut-off is at dilution 1/80.Results are expressed as reciprocal dilution titers (e.g., 80 meanspositive at dilution 1/80).

FIG. 20 illustrates the effect of lysozyme on the specificity of thehemagglutination assay on 200 Syphilis-negative blood bank serum samplesusing Tp17 reagent (R) or Tp17 reagent supplemented with recombinanthuman lysozyme (R+LyzR). The intensity of the agglutination in thenegative samples is represented by a number between 0 and 0.5. SD,standard deviation.

FIG. 21A illustrates the improved sensitivity of a second generationELISA syphilis assay in the presence of recombinant human lysozyme.

FIG. 21B illustrates the improved sensitivity of a third generationELISA syphilis assay in the presence of recombinant human lysozyme.

FIG. 22 shows a size-exclusion chromatographic separation of Tp17-Hisisoforms. Affinity purified Tp17-HIS is composed of three populations ofmolecules: monomeric Tp17-His (peak 3), dimeric Tp17-His (peak 2) andmultimeric Tp17-His (peak 1).

FIG. 23 shows a size-exclusion chromatographic separation of purified,recombinant human lysozyme (Ventria Biosciences, Sacramento, Calif.,USA). Recombinant human Lysozyme is composed of a single, highlyhomogenous population of molecules (peak 1).

FIG. 24 shows a size-exclusion chromatographic separation ofTp17-His/huLYS complexes. The chromatographic profiles corresponding tohuLYS complex with either monomeric Tp17-His (circles) or dimericTp17-His (diamonds) are represented. Peaks correspond to Tp17/huLYSprotein complexes (peaks 1 & 2) or monomer (huLys & Tp17) excesses(peaks 3 & 4).

FIG. 25 shows an SDS-PAGE electrophoretic separation of concentrated(5×) bacterial culture supernatant and the detection of secretedbacterial proteins by silver staining. The following pathogens wereanalyzed: Enterococcus faecalis (lane 2), Enterococcus faecium (lane 3),Staphylococcus aureus (lane 4), Streptococcus pneumoniae (lane 5),Streptococcus pyogenes (lane 6), Propionibacterium acnes (lane 7),Staphylococcus epidermidis (lane 8), Streptococcus agalactiae (lane 9).

FIG. 26 is a bar graph showing human lysozyme enzymatic activitymeasured in the absence, or in presence of (5×) concentrated bacteriaculture supernatant collected from the following eight bacterialspecies: Propionibacterium acnes, Streptococcus agalactiae,Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis,Streptococcus pneumoniae, Staphylococcus epidermidis and Enterococcusfaecium. The height of each bar indicates the detected enzymaticactivity. A lysozyme control is on the far left side of the figure.

FIG. 27 is a schematic diagram showing the principle and design of ahigh throughput screening assay for the identification of candidatecompounds capable of interfering with the formation of Tp17-likecomplexes with lysozyme. Step 1: Contacting a test sample withhorseradish peroxidase (POD)-labeled huLYS and a Tp17-like polypeptide.Step 2: incubation of the test sample at 37° C. Step 3: Extensivewashing, addition of POD substrate and signal detection.

FIGS. 28A and 28B are bar graphs showing the inhibition of commerciallyavailable, therapeutic preparations of chicken lysozyme by Tp17. FIG.28A shows the inhibition of bacteriolytic activity of Lizipaina®(Boehringer Ingelheim) by both GST-Tp17 and Tp17-His at various molarratios. FIG. 28B shows the inhibition of the bacteriolytic activity ofLizozima CHIESI (Laboratorio CHIESI) by GST-Tp17 at various molarratios. The height of each bar indicates the detected enzymaticactivity. A lysozyme control is on the far left side of the figure.

FIG. 29 is a bar graph showing that the bacteriolytic activity ofr-Lysozyme™ (Novagen, VWR International, Mollet del Vallés, Spain) wasnot susceptible to inhibition by GST-Tp17 or Tp17-HIS, while theantibacterial activity of human lysozyme was strongly inhibited. Theheight of each bar indicates the detected fluorescence. An r-Lysozyme™control and a human lysozyme (HuLys) control is present at the left ofeach experimental set.

FIG. 30 shows sequence alignments of the lysozyme binding/inhibitionsequences from Escherichia coli Ivy, Pseudomonas aeruginosa Ivy,Yersinia pestis Ivy, Treponema pallidum sp. pallidum Tp17 and the threeisoforms (APP770, APP751 & APP695) of the human Beta amyloid precursorprotein (β-APP). The Genbank accession number corresponding to eachpeptide sequence is indicated. The deduced consensus sequence isrepresented using the pattern syntax used in the PROSITE database.

FIG. 31 shows 3D views of the lysozyme binding/inhibition domains ofEscherichia coli Ivy, Pseudomonas aeruginosa Ivy and human β-APP.

FIG. 32 is a schematic diagram showing the domain organization of sAPPand Homo sapiens Beta amyloid precursor protein (β-APP).

FIG. 33 shows the detection of horse radish peroxidase (POD)-labeledhuLYS binding to immobilized sAPPα,. but not to immobilized sAPPα(304-612). 10 μg of both sAPPα and sAPPα (304-612) were spotted on anitrocellulose membrane and probed with POD-HuLYS diluted 1/1000.

FIGS. 34A and 34B are tables showing an alignment of lysozymebinding/inhibitor proteins, their Genbank accession numbers, and thehost species that the peptide is present in. These amino acid sequenceswere identified in silico using theCX(1,5)[KRH][AG][KRH]X(0,2)[KR]X(0,1)[EDQN]C (SEQ ID NO:178) peptidepattern (shown in FIG. 31) to search the Swiss-Prot and TrEMBL databasesusing the ScanProsite algorithm.

FIG. 35A is a schematic diagram showing a wild-type Tp17 polypeptide andmutant Tp17 CPA and KPA polypeptides. In TP17 CPA the histidine at aminoacid position 31 was replaced with alanine, and in Tp17 KPA, thehistidine at amino acid position 107 was replaced with alanine.

FIG. 35B is a table showing the inhibitory capacity of the mutant Tp17CPA and KPA polypeptides relative to the wild-type Tp17 polypeptide.

FIG. 36 is a Western blot showing proteins having molecular weights of45 kDA in each of lanes 1, 2, and 3 where lane 1 is the wild type Tp17,lane 2 CPA mutant, and lane 3 KPA mutant.

FIG. 37 shows an alignment of peptide sequences corresponding to humanlysozyme (huLYS) and chicken lysozyme (chkLYS), as well as huLYS andhuman Sperm Lysosyme-Like (SLLP1) protein. This alignment shows thatSLLP1 is closely related to both huLYS and chkLYS and support that Tp17may bind to SLLP1.

FIG. 38 is a schematic diagram showing that Tp17 may bind SLLP1 at thesperm surface and subsequently interfere with the fertilization process.

FIG. 39 shows a sequence alignment of lysozyme binding motifs present inthe E2 protein of some types of Hepatitis C virus.

FIG. 40 shows a sequence alignment of lysozyme binding motifs in the E2envelope glycoprotein of Hepatitis C virus genomes representative of thefollowing genomic groups: 1a, 1b, 1c, 2a, 2b, 2c, 2k, 3a, 3b, 3k, 4a,5a, 6a, 6b, 6d, 6g, 6h and 6k.

FIG. 41 shows the location of the lysozyme binding motif in the E2envelope glycoprotein of some Hepatitis C virus isolates with respect tohypervariable (HCR) region I and region II.

FIG. 42 is a schematic diagram showing a lysozyme binding motif in theectodomain of Hepatitis C virus E2 envelope glycoprotein.

FIG. 43A shows a sequence alignment of four amoebal lysozymes, AlyA,AlyB, AlyC, and AlyD.

FIG. 43B shows a sequence alignment of bacteriophage T4 lysozyme andfour amoebal lysozymes, AlyA, AlyB, AlyC, and AlyD.

DETAILED DESCRIPTION OF THE INVENTION

In general, the invention features compositions and methods relating tothe detection and treatment of subjects having a pathogen infection,such as Treponema pallidum. As described in more detail below, thisinvention is based, in part, on the discovery that a pathogen-expressedpolypeptide, Tp17, binds to and inhibits lysozyme, a host-expressedanti-microbial polypeptide. Because many pathogens express Tp17-relatedproteins, this method of inhibiting a host immune response is likely tobe conserved among a large number of pathogens. Accordingly, theinvention provides for methods of treating or preventing a pathogeninfection based on this observation. In addition, the invention providesfor improved diagnostic assays based on the detection of antibodies thatbind to a Tp17—lysozyme complex.

Treponema pallidum P17-Like Polypeptides

Compositions and methods of the invention comprise Treponema pallidumP17 (Tp17) polypeptide (Genbank Accession No. P29722; SEQ ID NO:31) andTp17-like polypeptides. These polypeptides share certain structuralsimilarities, as illustrated in FIGS. 1A, 1B, and 2, collectivelyreferred to herein as “Tp17-like polypeptides” and/or functionalsimilarities, such as the ability to bind lysozyme or inhibit lysozymeenzymatic or anti-microbial activity. At least one and possibly tworegions of Tp17 polypeptide (SEQ ID. NO:31) bind to lysozyme: thepolypeptide sequence VCPHAG (SEQ ID NO:5) at amino acid positions 28-33and the polypeptide sequence KAPHEK (SEQ ID NO:10) at amino acidpositions 114-119. FIGS. 1A and 2 provide an alignment of exemplaryTp17-like polypeptides from a number of bacterial, viral, parasitic, andmammalian species, along with their accession numbers. In particular,the alignment shown in FIG. 2 identifies an evolutionarily conservedconsensus sequence, Xaa_(n) Pro His Xaa_(n) (SEQ ID NO:1), which iscommon to all Tp17-like polypeptides listed in FIG. 2.

In an embodiment, a Tp17-like polypeptide comprises the amino acidconsensus sequence of Xaan Pro His Xaan (SEQ ID NO:1), wherein Xaa isany amino acid, is absent, or is a peptide bond, and n is at least one.

Referring now to FIGS. 1A and 1B, in another embodiment, the Tp17-likepolypeptide comprises the amino acid consensus sequence CS1: Cys Xaa₁Xaa₂ Xaa₃ Pro His Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Cys (SEQ ID NO:2), wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇,Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, or Xaa₁₃ is any amino acid, no aminoacid, or a peptide bond. In a preferred embodiment, Xaa₁ is Lys, Gly,Ile, Arg, Leu, Ala, Ser, Thr, Gln, Asn, Phe, Pro, no amino acid, or apeptide bond; Xaa₂ is Thr, His, Cys, Gln, Asn, Ile, Ser, Leu, Gly, noamino acid, or a peptide bond; Xaa₃ is His, Lys, no amino acid, or apeptide bond; Xaa₄ is Asn, Gly, Tyr, Leu, Ala, Cys, Glu, Thr, Arg, Met,Pro, Ile, Val, Phe, His, Gln, Lys, no amino acid, or a peptide bond;Xaa₅ is His, Arg, Asn, Leu, Ser, Lys, Glu, Gly, Pro, Ile, Thr, Cys, Trp,Val, Gln, Phe, Ala, Asp, no amino acid, or a peptide bond; Xaa₆ is Val,Cys, Ala, Asp, Ser, Pro, Gly, Glu, Lys, Leu, Gln, Ile, Tyr, Arg, Asn,Met, no amino acid, or a peptide bond; Xaa₇ is Asp, Glu, Ala, His, Val,Met, Pro, Lys, Arg, Cys, Asn, Gln, Phe, Leu, Ser, Trp, no amino acid, ora peptide bond; Xaa₈ is Gly, Asn, Ala, Ile, Arg, Glu, Lys, Thr, Tyr,Pro, Asp, Leu, Ser, no amino acid, or a peptide bond; Xaa₉ is Leu, Arg,Phe, Ile, Ala, Pro, Asn, Gln, Lys, Cys, Glu, Ser, no amino acid, or apeptide bond; Xaa₁₀ is Val, Thr, Asp, Glu, Tyr, Arg, Ala, Trp, no aminoacid, or a peptide bond; Xaa₁₁ is Pro, Asp, Lys, Asn, Gly, Thr, Ile, noamino acid, or a peptide bond; Xaa₁₂ is Ile, Val, Gly, Asp, Asn, Leu, noamino acid, or a peptide bond; and Xaa₁₃ is Asp, Glu, Cys, Thr, no aminoacid, or a peptide bond. Also, a preferred Tp17-like polypeptide thatdoes not strictly fit the consensus CS1 is the human immunodeficiencyvirus (HIV-1) Int protein with the related motif CSPEVGQMDC (SEQ IDNO:27) and the foot and mouth disease virus (FMDV) virus protein VP 1with the related motif TAPHRGLATLYNGDC (SEQ ID NO:26) (FIG. 1A).

Referring now to FIG. 2, in another embodiment, the Tp17-likepolypeptide comprises the amino acid consensus sequence CS2: Xaa₁ Xaa₂Pro His Xaa₃ Xaa₄ (SEQ ID NO:3), wherein Xaa₁ is Cys, Lys, Val, Ala, noamino acid or a peptide bond; Xaa₂ is Ala, Cys, or Lys; Xaa₃ is Ala,Asp, or Glu, and Xaa₄ is Cys, Gly, or Lys. In a preferred embodiment,the Tp17-like polypeptide comprises the amino acid sequence CCPHAG (SEQID NO:4), VCPHAG (SEQ ID NO:5), VAPHDC (SEQ ID NO:6), KAPHDK (SEQ IDNO:7), VKPHDG (SEQ ID NO:8), KKPHAK (SEQ ID NO:9), KAPHEK (SEQ IDNO:10), KKPHAC (SEQ ID NO:11), VAPHAG (SEQ ID NO:12), VKPHAK (SEQ IDNO:13), VKPHAC (SEQ ID NO:14), VAPHEG (SEQ ID NO:15), VKPHEK (SEQ IDNO:16), VCPHEK (SEQ ID NO:17), CKPHAG (SEQ ID NO:18), ACPHAG (SEQ IDNO:19), KCPHDC (SEQ ID NO:20), VKPHDK (SEQ ID NO:21), KKPHAG (SEQ IDNO:22), or CAPHEK (SEQ ID NO:23). In a preferred embodiment, theTp17-like polypeptide comprises Treponema pallidum P17 protein or HerpesSimplex Virus Type 2 glycoprotein J.

In an embodiment, the Tp17-like polypeptide, as defined herein, does notcomprise the Ivy protein. In another embodiment, the invention does notcomprise a protein comprising CKPHDC (SEQ ID NO:24).

In another embodiment, the Tp17-like polypeptides comprise consensussequence CS3, shared by Tp17 and Herpes simplex virus type 2 gJ protein,for example, corresponding to Xaa Cys Pro His Ala Gly (SEQ ID NO:25),wherein Xaa=Cys or Val.

FIG. 7 describes exemplary methods for targeted and randomidentification of polypeptide mutants.

Lysozyme Binding Motif Derived from Hepatitis C Virus

A Tp17-like polypeptide comprising the amino acid consensus sequence ofXaan Pro His Xaan is present in the E2 protein of some types ofHepatitis C virus. The alignment of exemplary Tp17-like polypeptidesfrom Hepatitis C virus is shown in FIG. 39. The alignment identifies aconsensus sequence of CPHRLSXaaC (SEQ ID NO:326), where Xaa is Ser orVal. Interestingly, further alignment of E2 envelope glycoproteins fromother types of Hepatitis C virus identifies lysozyme binding motifswhich do not strictly fit the consensus sequence of Xaa_(n) Pro HisXaa_(n) (SEQ ID NO:1). The sequences and the alignment of such lysozymebinding motifs are shown in FIG. 40. The alignment identifies aconsensus sequence of Cys Xaa₁ Xaa₂ Arg Xaa₃ Xaa₄ Xaa₅ Cys (SEQ IDNO:314). In preferred embodiments, Xaa1 is Pro, Ala, Val, or Ser; Xaa2is Gln or Glu; Xaa3 is Leu or Met; Xaa4 is Ser, Ala, or Gly; and Xaa5 isSer, Ala, Lys, or Cys. FIGS. 42 and 43 illustrate the location of thelysozyme binding motif in the E2 envelope glycoprotein and the relativeposition of the lysozyme binding motif with respect to hypervariable(HVR) region I and region II.

Lysozyme Molecules

Lysozyme causes the hydrolysis of bacterial cell walls. It is generallyfound in fluids contacting mucosal surfaces as well as other body fluidsand constitutes a defense mechanism against bacterial infections. Theenzyme cleaves the glycosidic bond between carbon number 1 ofN-acetylmuramic acid and carbon number 4 of N-acetyl-D-glucosamine. Invivo, these two carbohydrates are polymerized to form a cell wallpolysaccharide. Lysozyme has a multi-domain, mixed alpha and beta foldstructure, containing four conserved disulfide bonds.

Lysozyme also has a strong antiviral effect against herpes simplex virus(HSV) and HIV type 1 (HIV-1). The formation of syncytia in cellmonolayers infected with HSV is inhibited by hen egg-white lysozyme(Cisani et al. (1989) Microbios. 59:73-83). In addition, lysozymeenhances the anti-herpetic activity of glycyrrhizic acid in in vitroassays (Lampi et al. (2001) Antivir. Chem. Chemother. 12:125-131). Inaddition, anti-HIV-1 activity found in preparations of human chorionicgonadotrophin (hCG) has been attributed in part to lysozyme C (Lee-Huanget al. (1999) Proc. Natl. Acid. Sci. USA 96:2678-2681). In addition, anE. coli protein named Ivy was reported to bind and inhibit human andchicken lysozyme (Monchois et al. (2001) J. Biol. Chem.276:18437-18441). The interactions between lysozyme and such pathogenproteins and the mechanism of their action are not clear in the priorart. As described herein, the characterization of the interactionbetween Tp17 antigen and lysozyme provides, at least in part, anexplanation of how lysozyme interacts with pathogenic proteins andinhibits their activity.

Lysozyme levels are lower in patients suffering from HSV infectioncompared to healthy controls, suggesting that pathogen infection isassociated with a decrease in lysozyme production or an inhibition oflysozyme activity. The instant invention provides, at least in part, anexplanation of how lysozyme activity is inhibited by proteins producedby invading pathogenic viruses, suggesting a mechanism by whichpathogenic viruses evade lysis by lysozyme by producing an inhibitortherefor. The instant invention therefore provides compositions andmethods for relieving lysozyme inhibition in vitro and for theprevention or treatment of pathogen infection in vivo.

The compositions and methods of the invention comprise human lysozymeand polypeptides with sequence similarities thereto. FIGS. 3A and 3Bprovide an alignment of lysozyme polypeptides from a number of species,including duck, chicken, quail, guinea fowl, pheasant, peafowl, turkey,chachalaca, goat, sheep, cow, deer, pig, rat, mouse, dog, rabbit,monkey, grivet, rhesus monkey, baboon, marmoset, tamarin, squirrelmonkey, gorilla, human, orangutan, gibbon, colobus, langur, camel,possum, trout, flounder, fish, dog, horse, donkey, echidna, pigeon,hoatzin, anopheles, bombyx, cecropoia, silkmoth, trichoplusia, hornworm,and webworm. The alignment of the lysozyme proteins defines a lysozymeconsensus sequence of: Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa (SEQ ID NO: 28)Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa Xaa Xaa Thr Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly Xaa Xaa Gln Ile Asn XaaXaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa XaaXaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Ala LysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp Xaa Xaa XaaCys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa,

wherein Xaa is any amino acid or is absent. In an embodiment, thelysozyme consensus sequence comprises the sequence of“XkXXXrCelaXX$kXXgXdgyXgXs$X#WvClaXXESXXnTXatnXnXXXgStDYGifQIN (SEQ IDNO: 32) sXyWCndgktpXXXnXCX!XCsXL1Xd#itXaCAKk!vXdXXgXXaWvaWkXhCXgXdlsXyXXgCXXX”,where amino acid conservation is represented according to Rislercomparisons symbols (Risler et al. (1988) J. Mol. Biol. 204:1019). Inthat representation system, X represents any amino acid or is absent,upper case letters correspond to 100% amino acid conservation,lower-case letters correspond to a value of amino acid conservationcomprised between 50% and 90%. Moreover, conservative changes shared bymore than 50% of the sequences are represented using the followingsymbols: $ corresponds to leucine or methionine, ! corresponds toisoleucine or valine, and # corresponds to asparagine or aspartic acid.

The compositions and methods of the invention also comprisebacteriophage T4 lysozyme and polypeptides with sequence similaritiesthereto. FIG. 43A provides an alignment of lysozyme polypeptides fromamoeba, Dictyostelium discoideum, including AlyA (Genbank accessionnumber EAL69842.1 (SEQ ID NO:327)), AlyB (Genbank accession numberEAL69840.1 (SEQ ID NO:328)), AMyC (Genbank accession number EAL69841.1(SEQ ID NO:329)), and AlyD (Genbank accession number EAL70816.1 (SEQ IDNO:330)). The alignment defines a consensus sequence as illustrated inFIG. 43A. FIG. 43B provides an alignment of bacteriophage T4 lysozyme(gene E product, Genbank accession number P00720 (SEQ ID NO:331)), AlyA,AlyB, AlyC, and AlyD lysozymes. The alignment defines a consensussequence as illustrated in FIG. 43B. As shown in FIG. 43B, the consensussequence includes an N-terminal region defined by the following aminoacid sequence: Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa (SEQ ID NO: 322) Xaa XaaXaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Gly,

and a C-terminal region defined by the following amino acid sequence:Asp Ala Xaa Xaa Xaa Xaa Xaa Xaa (SEQ ID NO: 323) Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asnwherein Xaa is any amino acid or is absent.

In one embodiment, the N-terminal region comprises the following aminoacid sequence: Tyr Tyr Leu Thr Xaa Phe Cys Xaa (SEQ ID NO: 324) Xaa XaaXaa Ala Cys Gly Xaa Xaa Xaa Xaa Cys Ser Xaa Xaa Xaa Tyr Phe Thr Ala AspSer Gln Arg Phe Gly Cys Glywherein Xaa is any amino acid or is absent.

In one embodiment, the C-terminal region comprises the following aminoacid sequence: Asp Ala Gly Pro Xaa Xaa Xaa Val (SEQ ID NO: 325) Glu XaaXaa Ala Gly Xaa Xaa Ile Ile Asp Ala Ser Xaa Xaa Ile Cys Xaa Xaa Leu XaaGly Xaa Ser Ser Cys Gly Trp Ser Asp Xaa Xaa Xaa Ile Thr Ala Xaa Xaa XaaSer Xaa Xaa Asp Xaa Xaa Pro Xaa Xaa Xaa Pro Phe Asn Val Thrwherein Xaa is any amino acid or is absent.

In addition, as shown in FIGS. 43A and 43B, AlyD lysozyme polypeptidecontains a long Gly and Ser rich region separating the N-terminal regionfrom the C-terminal region. Without being tied to any particular theory,the existence of such a Gly-Ser rich region is likely to suggest thatthe N-terminal region and the C-terminal region may be structurallyindependent from each other.

Without being tied to any particular theory, the new consensus sequenceand possible new structural feature suggest that bacteriophage T4lysozyme, AlyA, AlyB, AlyC, and AlyD lysozymes may belong to a newfamily and may interact differently with Tp17-like polypeptides. As aresult, bacteriophage T4 lysozyme and similar lysozymes, including AlyA,AlyB, AlyC and AlyD lysozyme polypeptides, may not be inhibited byTp17-like polypeptides. Therefore, bacteriophage T4 lysozyme familypolypeptides can be used to prevent or treatment of pathogen infection.

Additional lysozyme sequences and alignments are disclosed in Müller etal. (2005) “A Dictyostelium Mutant with Reduced Lysozyme LevelsCompensates by Increased Phagocytic Activity,” J. Biol. Chem.,280:10435-10443, the disclosures of which are incorporated herein byreference in its entirety.

The term “lysozyme” or “lysozyme polypeptide” encompasses all homologs,mutants, full-length or fragments, substitutions and modificationsthereof, including deletions and additions thereto. In an embodiment, alysozyme polypeptide includes a mutation that decreases its ability tobind to or otherwise associate or interact with a Tp17-like polypeptide.As a result, the mutant lysozyme polypeptide is resistant to inhibitionby a pathogen-expressed Tp17-like polypeptide. Example 7 describesexemplary methods for targeted and random identification ofinhibition-resistant lysozyme mutants. Exogenous lysozyme is lysozymethat originates outside of, e.g., is not naturally occurring or occursin a significantly lower concentration in, the sample or Tp17-likepolypeptide preparation used in the methods of the invention. In anembodiment, exogenous lysozyme is lysozyme that is added to the sampleor to the Tp17-like polypeptide to make up the compositions and methodsof the invention. Tp17-like Polypeptide and Lysozyme Homologs, Mutantsand Derivatives

The invention also provides purified or recombinant variants ofTp17-like polypeptides or lysozyme polypeptides, including homologs,mutants, and derivatives. In some examples, recombinant mutantpolypeptides contain specific mutations (e.g., missense mutations,insertions, deletions, or nonsense mutations) that inhibit thebiological activity of the recombinant polypeptide relative to a nativeTp17-like polypeptide or lysozyme polypeptide. Particularly advantageousTp17-like mutant polypeptides bind lysozyme with an affinity that isequal to or greater than the affinity of a naturally-occurringpolypeptide, but fail to inhibit the anti-microbial activity oflysozyme, such as its ability to cleave a pathogen-expressedpolysacharide. Such Tp17-like mutant polypeptides are useful in treatingor preventing a pathogen infection.

Particularly advantageous mutant lysozyme polypeptides of the inventionare incapable of binding, or exhibit reduced binding to a Tp17-likepolypeptide relative to a native lysozyme (FIG. 3). Preferably, binding,e.g., binding affinity or quantity, is reduced by at least 5%, 10%, 25%,50%, 75%, 85%, 95%, or 100%. Other advantageous lysozyme polypeptidesretain the ability to bind a Tp17-like polypeptide, but such bindingdoes not inhibit the anti-microbial activity of the lysozymepolypeptide, such as its enzymatic activity.

Specific biological effects can be elicited by the use of a homolog,mutant, or derivative of limited function, e.g., with fewer side effectsor greater therapeutic potency, relative to the use of a reference or anaturally occurring (i.e., a wild-type) form of a Tp17-like polypeptideor lysozyme polypeptide. Mutants of a Tp17-like polypeptide or lysozymepolypeptide can be generated by mutagenesis, for example, by introducinga discrete point mutation(s), an insertion, a deletion or othermodifications and alterations. For instance, naturally occurring orlaboratory induced mutations can generate mutant Tp17-like polypeptidesor lysozyme polypeptides that retain substantially the same, or merely asubset, of the biological activity of the Tp17-like polypeptide orlysozyme polypeptide from which the mutant polypeptide was derived.Alternatively, antagonistic forms of a Tp17-like polypeptide or lysozymepolypeptide can be generated. Such antagonists inhibit the function of anaturally occurring Tp17-like polypeptide or lysozyme polypeptide, bycompetitively binding to a naturally occurring ligand.

The purified or recombinant homologs of Tp17-like polypeptides orlysozyme polypeptides of the present invention also include naturallyoccurring homologs of the wild-type Tp17-like polypeptides or lysozymepolypeptides that are naturally resistant to proteolytic cleavage.

Tp17-like polypeptides or lysozyme polypeptides may also be chemicallymodified to create Tp17-like polypeptide or lysozyme polypeptidederivatives, respectively, by forming covalent or aggregate conjugateswith other chemical moieties, such as glycosyl groups, lipids,phosphate, acetyl groups, and the like. Covalent derivatives ofTp17-like polypeptides or lysozyme polypeptides can be prepared bylinking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C-terminus of theprotein.

Modification of the structure of the Tp17-like polypeptides or lysozymepolypeptides (e.g., amino acid mutations) can be for such purposes asenhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivoshelf life and resistance to proteolytic degradation), orpost-translational modifications (e.g., to alter phosphorylation patternof protein). Such modified or mutant Tp17-like polypeptides or lysozymepolypeptides, when designed to retain at least one activity of thenaturally-occurring form of the Tp17-like polypeptides or lysozymepolypeptides, or to produce specific antagonists thereof, are consideredfunctional equivalents of the Tp17-like polypeptides or lysozymepolypeptides described in more detail herein. Such modified Tp17-likepolypeptides or lysozyme polypeptides can be produced, for instance, byamino acid substitution, deletion, or addition. The substitutionalvariant may be a substituted conserved amino acid or a substitutednon-conserved amino acid.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e., isosteric and/or isoelectricmutations) do not have a major effect on the biological activity of theresulting molecule. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains.Genetically encoded amino acids can be divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. In similar fashion,the amino acid repertoire can be grouped as (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine,alanine, valine, leucine, isoleucine, serine, threonine, with serine andthreonine optionally be grouped separately as aliphatic-hydroxyl; (4)aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine,glutamine; and (6) sulfur-containing=cysteine and methionine. Whether achange in the amino acid sequence of a peptide results in a functionalTp17-like polypeptide or lysozyme polypeptide homolog (e.g., functionalin the sense that the resulting polypeptide mimics or antagonizes thewild-type form) can be readily determined by assessing the ability ofthe variant peptide to produce a response in an assay or in cells in afashion similar to the wild-type protein, or competitively inhibit sucha response. Polypeptides in which more than one replacement has takenplace can readily be tested in the same manner. Assays and reagentsuseful for testing such homologs are exemplified throughout thespecification.

The term “polypeptide” thus includes naturally occurring peptides orproteins, as well as synthetic or recombinantly produced peptides orproteins. The polypeptide may encompass amino acid chains of any length,wherein the amino acid residues are linked by covalant peptide bonds.However, peptidomimetics of such polypeptides wherein amino acids and/orpeptide bonds have been replaced by functional analogs are alsoencompassed by the invention. In accordance with the invention, an aminoacid encompasses a non-naturally occurring amino acid analog.

This invention further contemplates a method for generating sets ofcombinatorial mutants of Tp17-like polypeptides or lysozyme polypeptidesas well as truncation mutants, and is especially useful for identifyingpotential variant sequences (e.g., homologs). The purpose of screeningsuch combinatorial libraries is to generate, for example, novel mutantTp17-like polypeptides or mutant lysozyme polypeptides that can act aseither agonists or antagonist, or alternatively, possess novelactivities altogether. Thus, combinatorially-derived mutant polypeptidescan be generated that have an increased potency relative to anaturally-occurring (wild-type) form of the polypeptide.

In another embodiment, the invention features isolated Tp17-likepolypeptides or lysozyme polypeptides, preferably substantially purepreparations, e.g., of body-fluid derived or recombinantly producedpolypeptides. The Tp17-like polypeptides or lysozyme polypeptides cancomprise full length polypeptides or can comprise smaller fragmentscorresponding to one or more particular motifs/domains, or fragmentscomprising at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, about20, about 25, about 50, about 75, about 100, about 125, about 148, aminoacids in length, for example.

Peptides may be produced by direct peptide synthesis using solid phasetechniques (e.g., Stewart et al. (1969) Solid Phase Peptide Synthesis,WH Freeman Co. San Francisco; Merrifield (1963) J. Am. Chem. Soc.85:2149-2154). In vitro protein synthesis may be performed by in vitrocoupled transcription and translation, for example using a TnT®(Promega, Madison, Wis.) or RTS (Roche Applied Science, Barcelona,Spain) kits or using an automated approach, for example, using anApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City,Calif.) according to the manufacturers instructions. Peptides may bemade by one or more methods and chemically joined to produce a fulllength molecule.

The characterization of the lysozyme binding site on Tp17 and theconsensus sequence of that binding site that is shared by a number ofpathogen-derived Tp17-like polypeptides can be exploited to provideinhibitors of lysozyme/Tp17-like polypeptide binding and methods ofmaking and using them as diagnostics (e.g., probes), prophylactics, ortreatments for a number of pathogenic diseases or diseases characterizedby altered lysozyme activity. Also, the invention provides natural andgenetically modified variants of lysozymes, including homologs, mutants,and derivatives and methods of making them, that are unable to bind toTp17-like polypeptides, which retain other lysozyme functions, such aspeptidoglycan binding and hydrolysis for example, thereby escapinginhibition by the Tp17-like polypeptides. These non-wildtype lysozymes,including homologs, mutants and derivatives can be used to treat orprevent many pathogenic diseases, including syphilis, AIDS, and genitalherpes.

The invention also provides Tp17-like mutant polypeptides that havealtered lysozyme binding sequences, and mutant lysozyme polypeptidesthat have altered Tp17-like polypeptide binding sequences. Preferably,these mutant polypeptides exhibit altered binding affinities.Particularly preferred are Tp17-like mutant polypeptides having anincrease (e.g., 5%, 10%, 25%, 50%, 75%, or 100%) in their binding tolysozyme relative to a native Tp17-like polypeptide. Such mutantpolypeptides compete with a native Tp17-like polypeptide for binding tolysozyme. Most preferred are Tp17-like mutant polypeptides that bind tolysozyme with at least native affinity, but that fail to inhibit alysozyme anti-microbial activity, such as an enzymatic activity.Particularly preferred lysozyme mutant polypeptides have altered bindingsites that reduce (e.g., 5%, 10%, 25%, 50%, 75%, or 100%) or eliminatebinding to a Tp17-related polypeptide. Other preferred lysozyme mutantpolypeptides bind a Tp17-related polypeptide, but such binding does notreduce or eliminate lysozyme anti-microbial activity.

Other alterations in a Tp17-related polypeptide or in a lysozymepolypeptide alter in vitro or in vivo function, for example, as effectedby site-directed mutagenesis or combinatorial biochemistry. For example,lysozyme variants that have lost the ability to bind to peptidoglycanbut retain the ability to bind to Tp17-like polypeptide are provided.

The invention further provides Tp17-like polypeptides that can be usedto make fusion polypeptides with a polypeptide of interest, which can beinexpensively affinity purified using lysozyme (e.g., that is bound to asolid support, such as a resin, bead, well, chip, column, gel, membrane,or filter device).

The sequence information provided by the instant invention also providesspecific probes for the identification of Tp17-like polypeptides using alysozyme as a probe, or identification of lysozyme using a Tp17-likepolypeptide as a probe. In addition, antibodies can be raised tosequences that define these binding sites using standard methods.

Methods for Improving Pathogen Infection Detection Methods

The instant invention also provides methods and compositions forimproved detection of anti-pathogen antibodies in a sample, for example,as diagnosis of pathogen infection. While examples of such diagnosticmethods may specifically relate to syphilis, one skilled in the art willunderstand that such methods are generally useful for detecting animmune response against virtually any pathogen that produces a Tp17-likepolypeptide. The diagnostic methods of the invention involve thedetection of an anti-pathogen antibody in a sample using reagents thatinclude, among other things, lysozyme. Example 8 provides a descriptionof an exemplary syphilis assay for anti-Tp17-like polypeptideantibodies. The above and other methods are discussed in more detailbelow.

Without being tied to any particular theory, it is likely that asyphilitic subject generates an immune response not only against anisolated Tp17 protein, but also against the Tp17 polypeptide/lysozymecomplex. Contacting a biological sample from such a subject withlysozyme, allows for the detection of antibodies that recognize theTp17/lysozyme complex, and increases the sensitivity of the syphilisdiagnostic assay. Similarly, any pathogen diagnostic method thatinvolves detection of an antibody that recognizes a Tp17-likeprotein/lysozyme complex is enhanced by the inclusion of lysozyme in theassay reagents. Preferably, the addition of lysozyme to an assay reagentincreases the sensitivity of the diagnostic method by at least 5%, 10%,25%, 50%, 75%, or even by as much as 85% or 95% relative to standarddiagnostic methods.

Compositions and Methods for Detecting Anti-Pathogen Antibodies

The invention provides compositions and methods for detectinganti-pathogen antibodies in a sample that are indicative of the presenceof, infection by, or immune response (e.g., humoral immune response) toa bacterial pathogen, such as, for example, Treponema pallidum,Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Yersiniapestis, Shigella flexnerii, Treponema denticola, Vibrio cholerae, Vibriovulnificus, Vibrio parahemolyticus, Chlamydia pneumoniae, Staphylococcusaureus, Staphylococcus epidermidis, Streptococcus pneumoniae,Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Enterococcus faecalis, Bordetella bronchiseptica, Bordetella pertussis,Bordetella parapertussis, Helicobacter hepaticus, Salmonellatyphimurium, Ralstonia solanacearum, Xanthomonas campestris, Pseudomonassyringae, Pasteurella multocida, Brucella melitensis, Brucella suis,Mycobacterium tuberculosis, Campylobacter jejuni, Helicobacter spp., andAgrobacterium tumefaciens, Moraxella catarrhalis, Neisseriameningitidis, Neisseria gonorrhoeae, Mycoplasma pneumoniae, Legionellasp., Haemophilus influenzae, Haemophilus ducreyii, Propionobacteriumacnes, Listeria monocytogenes. In addition, the composition is used todetect the presence of, infection by, or immune response (e.g., humoralimmune response) to a viral pathogen, such as, for example, Coxsackievirus, Herpes Simplex Virus type 2, Influenza A virus, TACARIBE virus,Bluetongue virus, Chimpanzee cytomegalovirus, Hepatitis B, Hepatitis Cvirus, Human cytomegalovirus (HCMV), Human papilloma virus, Denguevirus, eastern equine encephalitis virus, western equine encephalitisvirus, Venezuelan equine encephalitis virus, Foot and Mouth Disease(HMD), Human immunodeficiency virus, Rubella virus, cattle plague virus,rabies virus, and LdMNPV. Further, the composition is used to detect thepresence of, infection by, or immune response (e.g., humoral immuneresponse) to a parasite, such as, for example, Entamoeba histolytica,Plasmodium falciparum, Plasmodium ovale, Emeiria tenella, Eimeriaacervulina, Giardia lamblia, Plasmodium yoelii and pathogens carried byAnopheles gambiae. In addition, the composition is used to detect thepresence of, infection by, or immune response (e.g., humoral immuneresponse) to Mycobacterium paratuberculosis, Ebola virus, Rift ValleyFever virus, Severe Acute Respiratory Syndrome (SARS) virus, Small Poxvirus, Bacillus anthracis, Leishmania Spp., mycoplasma, rickettsia,fungi, or yeast. Such antibodies are identified, for example, by anassay that detects antibody binding to a Tp17-like polypeptide, alysozyme polypeptide, a TP 17-like polypeptide/lysozyme polypeptidecomplex, or a fragment thereof.

The Tp17-like polypeptide or lysozyme polypeptide may comprise anaffinity tag, such as, for example, glutathione S-transferase (GST),6-histidine tail (HIS), maltose binding protein, elastin-like peptide,or a Strep-Tag (IBA, Goettingen, Germany). The Tp17-like polypeptide,lysozyme, and/or Tp17-like polypeptide-lysozyme complex may be attachedto a solid support such as a resin, bead, well, chip, column, gel,membrane, matrix, plate, or filter device. The use of a multi-well plateor a microchip is useful for the large scale testing of numerous samplesor for testing a single sample in duplicate or for the presence of anumber of pathogenic agents.

In one embodiment, the method of the invention comprises detectingbinding, if present, of anti-pathogen antibodies present in the sampleto (i) the Tp17-like polypeptide alone; (ii) the lysozyme alone; or(iii) the Tp17-like polypeptide-lysozyme complex. In one embodiment, themethod comprises the steps of (a) contacting a sample with a Tp17-likepolypeptide under conditions that permit binding of an antibody in thesample to the Tp17-like polypeptide; and (b) contacting the sample witha substantially pure, exogenous lysozyme under conditions that permitbinding of an antibody to the lysozyme. The Tp17-like polypeptide andlysozyme may be bound to each other or otherwise associated such that anantibody can bind to both the Tp17-like polypeptide and lysozyme, e.g.,a complex of the Tp17-like polypeptide and lysozyme. Binding of theantibody to the Tp17-like polypeptide, lysozyme, or their complex mayoccur at 15-25° C., or any temperature that allows binding of theantibody to the Tp17-like polypeptide and/or lysozyme.

In a preferred embodiment, the assay is an agglutination assay, such as,for example, a hemagglutination assay performed as described in Examples8 and 9. The carrier particles used in the agglutination assay may be,for example, red blood cells, protein aggregate particles, polymericparticles, inorganic particles, paramagnetic particles, or yeast cells.In a preferred embodiment, hemagglutination is enhanced by at leastabout 0.1 to about 20 fold, about 0.3 to about 10 fold, or about 1 toabout 10 fold, for example, preferably at least about 3-fold, by theaddition of lysozyme. The lysozyme may be from the same species as thesubject from which the assay sample is taken (e.g., human). The lysozymeis added to the Tp17-like polypeptide reagent, the dilution buffer, orany of the reagents or vessels used in the assay and may be added at anytime, once or in installments, e.g., before, during or after the samplehas contacted the remainder of the reagents or vessels of the assay. Ina particular embodiment, exogenous lysozyme is present in aconcentration range of about 1 femtogram/ml to about 999 milligrams/ml.,lysozyme is present in a concentration range of about 1 nanogram/ml toabout 999 micrograms/ml (e.g., 1, 10, 25, 50, 100, 250, 500, or 1000nanograms or micrograms/ml).

In another embodiment, anti-pathogen antibodies are detected using anELISA assay. Methods for carrying out ELISA assays are well known in theart. Briefly, for detecting the presence of antibodies to Tp17-likepolypeptides in a sample, for example, a solid phase, such as an ELISAplate, is coated with a Tp17-like polypeptide alone, a combination ofTp17-like polypeptide and lysozyme, or lysozyme alone, in separate wellson a plate. After washing, a sample that may contain an anti-Tp17-likepolypeptide antibody, an anti-Tp17-like polypeptide-lysozyme complexantibody, and/or anti-lysozyme antibody is added to the wells. Thesample may be applied to several wells of the ELISA plate, and detectedvia direct labeling (if appropriate), by using a secondary bindingpartner for the anti-Tp17-like polypeptide antibody such as a rabbitanti-human IgG that has a detectable label, or by using a tertiaryantibody or detection reagent (e.g., streptavidin-biotin or labeledprotein A or protein G). Alternatively, detection can also be achievedusing labeled Tp17-like polypeptide. If the specific antibodies aredifferentially labeled, detection of more than one antibody can occur inthe same sample, for example in the same well of the ELISA plate. Thiscan be accomplished through the use of labels that produce distinctsignals that can be independently quantified, for example, by using dyeswith different UV absorption maxima. The ELISA values for the variouslytreated wells can be compared to determine the presence of antibodies toTp17-like polypeptide alone, Tp17-like polypeptide-lysozyme complex,and/or lysozyme alone. Any of the immunoassays described herein may beused in the practice of detecting antibodies to Tp17-like polypeptideand the Tp17-like polypeptide-lysozyme complex by the use of lysozymewhich creates, either in the liquid or solid phase, Tp17-likepolypeptide-lysozyme complexes that can bind to their cognateantibodies, according to standard methods.

Not to be limited to a particular theory, the assay is likely enhancedby the addition of lysozyme because the lysozyme forms complexes withthe Tp17-like polypeptides, thereby forming substrate for binding withantibodies that bind only to the Tp17-lysozyme complex and not toTp17-like polypeptide alone. Thus, by adding lysozyme to the assay,antibodies directed to the Tp17-like polypeptide alone, the lysozymealone or the complex are detected in the sample (FIG. 4).

In an embodiment of the invention, the assay can be accomplished byusing a natural purified or recombinant lysozyme that does not havepeptidoglycan binding activity (e.g., a lysozyme mutant) so that bindingof the lysozyme to the Tp17-like polypeptide is enriched. This selectivebinding can be achieved, for example, by site-directed mutagenesis oflysozyme, or other methods known in the art, for example, as describedin Example 7.

In another embodiment, the lysozyme molecule can be altered to removeits ability to bind to a Tp17-like polypeptide, so that the resultingmutant is resistant to inhibition by a Tp17-like polypeptide. Suchmutants may display an enhanced antimicrobial activity against pathogensequipped with Tp17-like polypeptide virulence factors. Mutationslocalized in the lysozyme coding sequence, selected so that acetylmuramidase and/or antimicrobial activities are not affected, destabilizethe lysozymes/inhibitor interaction and confer resistance to inhibitionby pathogen proteins such as the T. pallidum Tp17 antigen or members ofthe Ivy family. Such mutant lysozymes represent novel and interestingcompositions for the treatment of infectious diseases. In addition,given their improved potency and spectrum of activity, they are superiorto the chicken lysozyme presently used in commercially availablemedicinal preparations.

Compositions and Methods for Inhibiting Lysozyme Activity In Vitro

In another aspect, the invention provides compositions and methods forreducing lysozyme activity in a sample by contacting a sample with aTp17-like polypeptide, or fragment thereof, where the Tp17-likepolypeptide binds to and inhibits lysozyme activity in the sample. Thetreatment is useful for any sample that requires inactivation orneutralization of lysozyme, such as, for example, a preparation of apharmaceutical fluid for human administration, a cell culture fluid, afood, a medicine, water, or other fluid or agent for ingestion, animplant, a graft, or any other preparation in which lysozyme is notdesired or is harmful. The compositions and methods of the invention aresuitable for small or large scale treatment of water supplies, forexample. The compositions and methods may be used to inhibit lysozymeson a material that is supposed to be free of lysozyme contamination, orfor which inhibition of lysozyme activity is desired, such as abiological sample, culture vessel, cuvette, swab, clinical diagnosticapparatus or assay material, medical instrument, cell culture, throataspirate, cerebospinal fluid sample, or hemoculture. In someembodiments, the Tp17-like polypeptide or lysozyme binding fragment isfixed to a solid support and the lysozyme present in the sample is boundto the TPI 7-like polypeptide.

In an embodiment, material from the forestomach of a ruminant animal maybe tested or treated using the methods and compositions of theinvention. This material includes bacteria, yeast, fungus, and protozoancells, for example. This flora controls the degradation and theassimilation of nutrients in ruminants. In an embodiment, the inhibitionof lysozyme by a Tp17-like polypeptide, or the inhibition of a Tp17-likepolypeptide by lysozyme, may alter the assimilation rate of nutrientsand be advantageous in certain circumstances.

The sample may also be a bacterial sample, such that the recovery of apathogen extract is enhanced by inhibiting cell lysis.

In addition, the invention is useful for identifying a bacteria bydetermining the extent to which lysozyme in the presence of a Tp17-likepolypeptide of predetermined sequence can inhibit bacterial cell lysis.For example, the methods of the invention may be used to distinguishbetween HSV-1 infection and HSV-2 infection (see Example 6).

Method for Reducing Lysozyme Activity In Vivo

The identification of the lysozyme binding motif on Tp17-likepolypeptides also provides compositions and methods (e.g., therapeuticor prophylactic, including vaccine) for inhibiting, inactivating orneutralizing lysozyme activity in an animal, such as a mammal, bird orfish. Tp17-like polypeptides comprising the lysozyme binding motif canbe used to block Tp17-like polypeptide binding to lysozyme. Theinvention therefore provides a method for reducing lysozyme activity inan animal by administering an effective amount of a Tp17-likepolypeptide. Such a method can be used to treat or prevent diseases suchas cancer, infectious diseases, inflammatory diseases, Alzheimer'sdisease, renal amyloidosis, leukemia, Crohn's disease, and allergy.

Methods for Detecting Pathogens and Tp17-Like Polypeptide Ligands

The compositions and methods of the invention may also be used fordetecting a pathogen in a sample. Compositions comprising a ligandcapable of binding to a Tp17-like polypeptide, such as lysozymepolypeptide or a molecule that can compete with lysozyme binding, may beused. Alternatively, the ligand may be a monoclonal antibody, apolyclonal antibody, or an Fab fragment that binds to the Tp17-likepolypeptide.

The ligands may be detected directly, e.g., linked to a detectionmolecule, such as a fluorophore, a fluorescent protein, chromophore,radioactive moiety, luminiferous moiety or enzymatically active reporteror label. Exemplary detection molecules are well known in the art, forexample, fluorescein conjugates, horseradish peroxidase conjugates,alkaline phosphatase conjugates and isoluminol conjugates. In thisembodiment of the invention, a sample is contacted with lysozyme or withanother Tp17-like ligand under conditions that permit binding of theligand to a pathogen or a pathogen polypeptide and the binding of thelysozyme to the pathogen or pathogen polypeptide, if present, isdetected. Such binding is indicative of the presence of the pathogen orpathogen polypeptide in the sample.

The compositions and methods of the invention are also useful fordetecting the presence of lysozyme in a sample. A sample is contactedwith a Tp17-like polypeptide and binding of the Tp17-like polypeptide tothe lysozyme in the sample is detected.

Kits

The compositions and methods of the invention may be embodied indiagnostic or therapeutic kits containing at least one of a Tp17-likepolypeptide, lysozyme, and a ligand therefore. Reagents necessary oruseful for the administration, assay, or purification of the Tp17-likepolypeptide or lysozyme polypeptide may also be included in the kit,such as reaction vessels (e.g., comprising a solid support, resin, bead,well, chip, column, gel, membrane, and filter device), controlstandards, or instruction manuals. Such diagnostic or therapeutic kitsare useful for the diagnosis or treatment of a pathogen infection, suchas syphilis or herpes, or for Alzheimer's disease.

Fusion Peptides and Affinity Chromatography

The Tp17-like polypeptides, or fragments thereof, of the invention areuseful as affinity tags for use in affinity chromatography usinglysozyme polypeptides, for example, that are linked to a solid support,such as a column. The invention therefore provides recombinant fusionproteins (e.g., Tp17 fusion proteins) comprising a first polypeptidesequence linked by a peptide bond to a second polypeptide sequence,wherein the first polypeptide sequence comprises a Tp17-like polypeptideand the second polypeptide sequence comprises an amino acid sequence ofinterest. In an embodiment, the first polypeptide sequence is notnaturally linked to the second polypeptide sequence. The Tp17-likepolypeptide acts as a purification cassette that is used to bind thepolypeptide of interest to the lysozyme affinity column. Thepurification cassette can be positioned anywhere on the molecule, e.g.,at the N terminus or the C terminus of the polypeptide of interest.Alternatively, the purification cassette can be positioned between the Nterminus and the C terminus. The recombinant fusion polypeptide isprepared by linking a first polypeptide sequence by a peptide bond to asecond polypeptide sequence, wherein the first polypeptide sequencecomprises a Tp17-like polypeptide purification cassette and the secondpolypeptide sequence comprises an amino acid sequence of interest. TheTp17 fusion protein is then used in methods for purifying thepolypeptide of interest by contacting a sample comprising the Tp17fusion protein with lysozyme under conditions that permit the Tp17-likepolypeptide to bind to lysozyme. The complex formed is then washed andthe Tp17 fusion protein is eluted from the lysozyme and the protein ofinterest may be isolated from the purification cassette according tomethods well known in the art. In one embodiment, the T17-likepolypeptide fragment is a 5, 10, 15, 20, 25, 50, 75, or 100 amino acidfragment that comprises at least a lysozyme-binding consensus sequence(e.g., SEQ ID NO:1).

Methods of Treating or Preventing Other Diseases

Lysozyme polypeptides are provided that do not bind to at least someTp17-like polypeptides (e.g., inhibition of lysozymes functions) due tothe presence of mutations. These mutations are targeted to residues thatdo not affect the catalytic activity of lysozyme. Such altered lysozymepolypeptides are made by standard methods (e.g., by site-directed orrandom mutagenesis as described herein). Such lysozymes are useful foravoiding inhibition by Tp17-like polypeptides and can be used to preventor treat a pathogen infection. The invention also provides methods forinhibiting pathogen infection by administering an effective amount of alysozyme polypeptide that cannot bind to Tp17-like polypeptides.

The invention also provides methods for identifying a reagent thatinhibits the binding of a lysozyme inhibitor such as Tp17-likepolypeptide to the E53 of a lysozyme (numbering refers to thefull-length human lysozyme sequence deposited under accession numberNP_(—)000230 (SEQ ID NO:302)). In a preferred embodiment, the reagent isa polypeptide, chemical, drug, antibody, nucleic acid, aptamer, or PNA.The inhibitor is, for example, any such molecule that prevents thebinding of Tp17-like polypeptides to E53 of lysozyme.

Screening Assays

As discussed above, binding of a Tp17-like polypeptide to a lysozymepolypeptide may inhibit lysozyme anti-microbial activity. Based on thisdiscovery, compositions of the invention are useful for thehigh-throughput low-cost screening of candidate compounds to identifythose that increase or decrease binding between a Tp17-like polypeptideand a lysozyme polypeptide. Any number of methods are available andcontemplated by this invention for carrying out screening assays toidentify new candidate compounds that modulate the binding of aTp17-like polypeptide and a lysozyme polypeptide.

In one embodiment, candidate compounds are screened for those thatspecifically bind a Tp17-like polypeptide or a lysozyme polypeptide. Theefficacy of such a candidate compound is dependent upon its ability tointeract with such a polypeptide or a functional equivalent thereof.Such an interaction can be readily assayed using any number of standardbinding techniques and functional assays (e.g., those described inAusubel et al., supra). In one embodiment, a candidate compound may betested in vitro for its ability to specifically bind a polypeptide ofthe invention. In another embodiment, a candidate compound is tested forits ability to enhance the biological activity of a lysozyme polypeptidedescribed herein, such as a Tp17-like polypeptide or a lysozymepolypeptide. The effect of the biological activity of the candidatecompound on the Tp17-like polypeptide and/or the lysozyme polypeptide isassayed using any standard method, known to the skilled artisan, such asthe assay described in Example 3.

In one particular example, a candidate compound that binds to aTp17-like polypeptide or a lysozyme polypeptide is identified using achromatography-based technique. For example, a recombinant polypeptideof the invention may be purified by standard techniques from cellsengineered to express the polypeptide (e.g., those described herein) andmay be immobilized on a column. A solution of candidate compounds isthen passed through the column, and a compound that specificallyinteracts with a Tp17-like polypeptide or a lysozyme is identified onthe basis of its ability to bind to the polypeptide and be immobilizedon the column. To isolate the compound, the column is washed to removenon-specifically bound molecules, and the compound of interest is thenreleased from the column and collected. Similar methods may be used toisolate a compound bound to a polypeptide microarray, for example, anarray containing a plurality of Tp17-like lysozyme binding motifs.Compounds isolated by this method (or any other appropriate method) may,if desired, be further purified (e.g., by high performance liquidchromatography). In addition, these candidate compounds may be testedfor their ability to modulate binding between a Tp17-like polypeptideand a lysozyme polypeptide. Binding may be altered, for example, byincreasing or decreasing the number of complexed molecules, by alteringthe binding affinity, by altering the probability that a complex willform, or by competing with one or both molecules for binding. In otherembodiments, the compound is assayed for its ability to increaselysozyme anti-microbial activity. In yet other embodiments, the compoundis assayed for its ability to modulate the interaction between apolypeptide that contains a CX(1,5)[KRH][AG][KRH]X(0,2)[KR]X(0,1)[EDQN]C(SEQ ID NO:178) consensus motif (such as a β-amyloid precursor protein)and a lysozyme polypeptide.

Compounds isolated by this approach may be used, for example, astherapeutics to treat a pathogen infection, a lysozyme disorder, orAlzheimer's disease in a human patient. Compounds that are identified asbinding to a polypeptide of the invention with an affinity constant lessthan or equal to 10 mM are considered particularly useful in theinvention. Alternatively, any in vivo protein interaction detectionsystem, for example, any two-hybrid assay may be utilized. Potentialagonists and antagonists include organic molecules, peptides, peptidemimetics, polypeptides, nucleic acids, and antibodies that bind to anucleic acid sequence or polypeptide of the invention, such as aTp17-related polypeptide or a lysozyme polypeptide. For patient having adisorder characterized by Tp17-like polypeptide inhibition of lysozyme(e.g., a pathogen infection), compounds that inhibit Tp17-likepolypeptide binding to lysozyme or that enhance a lysozymeanti-microbial activity or enzymatic activity are particularly useful.For patient having a disorder characterized by excessive lysozymeactivity, a compound that binds to lysozyme and inhibits its activity,or that enhances binding between a Tp17-like polypeptide and lysozymeare particularly useful.

DNA sequences encoding the Tp17-like polypeptides and lysozymepolypeptides listed herein may also be used in the discovery anddevelopment of a therapeutic compound for the treatment of patientshaving a pathogen infection, lysozyme disorder, or Alzheimer's disease.The encoded proteins, upon expression, can be used as targets for thescreening of drugs. Optionally, compounds identified in any of theabove-described assays may be confirmed as useful in an in vivo assayfor compounds that modulate binding between a Tp17-like polypeptide anda lysozyme polypeptide or that modulate the activity of a lysozymepolypeptide. Small molecules of the invention preferably have amolecular weight below 2,000 daltons, more preferably between 300 and1,000 daltons, and most preferably between 400 and 700 daltons. It ispreferred that these small molecules are organic molecules.

Test Compounds and Extracts

In general, compounds capable of modulating the binding of a Tp17-likepolypeptide to a lysozyme polypeptide are identified from largelibraries of both natural product or synthetic (or semi-synthetic)extracts or chemical libraries or from polypeptide or nucleic acidlibraries, according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s) of the invention. Compounds used in screens may includeknown compounds (for example, known therapeutics used for other diseasesor disorders). Alternatively, virtually any number of unknown chemicalextracts or compounds can be screened using the methods describedherein. Examples of such extracts or compounds include, but are notlimited to, plant-, fungal-, prokaryotic- or animal-based extracts,fermentation broths, and synthetic compounds, as well as modification ofexisting compounds. Numerous methods are also available for generatingrandom or directed synthesis (e.g., semi-synthesis or total synthesis)of any number of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Furthermore, if desired, any library or compound is readily modifiedusing standard chemical, physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their activity should be employed wheneverpossible.

When a crude extract is found to modulate the binding between aTp17-like polypeptide and a lysozyme polypeptide, further fractionationof the positive lead extract is necessary to isolate chemicalconstituents responsible for the observed effect. Thus, the goal of theextraction, fractionation, and purification process is the carefulcharacterization and identification of a chemical entity within thecrude extract that alters the binding between a Tp17-like polypeptideand a lysozyme polypeptide. Methods of fractionation and purification ofsuch heterogenous extracts are known in the art. If desired, compoundsshown to be useful as therapeutics for the treatment of a pathogeninfection or a lysozyme disorder are chemically modified according tomethods known in the art.

Antibody Preparation

Suitable monoclonal antibodies for use in the immunoassays of theinvention may be prepared by standard hybridoma methods, usingdifferential binding assays to ensure that the antibodies are specificfor a Tp17-like polypeptide, lysozyme, a Tp17-like polypeptide-lysozymecomplex or other antigen of interest and do not show cross-reactivity,or show limited cross-reactivity between the related proteins.Alternatively, suitable monoclonal antibodies may be prepared usingantibody engineering methods such as phage display. Methods forobtaining highly specific antibodies from antibody phage displaylibraries are known in the art, and several phage antibody libraries arecommercially available from, for example, MorphoSys (Martinsried,Germany), Cambridge Antibody Technology (Cambridge, UK) and Dyax(Cambridge, Mass.). Suitable phage display methods are described, forexample, in U.S. Pat. Nos. 6,300,064 and 5,969,108, which are herebyincorporated by reference in their entirety. See also, for example“Antibody Engineering,” McCafferty et al. (Eds.)(IRL Press 1996) andreferences therein. Phage display antibody methods can use libraries ofantibodies in the Fab or scFv format. Once the antibody heavy and lightchain genes are recovered from the phage antibodies, antibodies in anysuitable format may be prepared (e.g., whole antibodies, Fab, scFv,etc.).

Other antibody preparations may also be used, for example, Camelidantibodies, which contain only heavy immunoglobulin chains. See, forexample, Muyldermans et al. (2001) J. Biotechnol. 74:277-302 andreferences therein.

Alternatively, polyclonal antibody preparations may be used fordetection of antigens such as Tp17-like polypeptides, lysozyme, or otherantigen of interest. Phage display methods also can be used to preparereproducible populations of polyclonal antibodies. For example, anantibody library can be exhaustively depleted of clones that cross-reactby absorption on other antigens bound to a solid surface, and thenpanned over a solid surface to identify antibodies that bind to theantigen of interest. The resulting population of clones can also bedepleted of cross-reactive clones by absorption over surfaces bearingirrelevant proteins, such as bovine serum albumin, etc., using methodswell known in the art. This results in identification of a population ofantibodies that specifically bind to an antigen of interest.

Polyclonal antibodies specific for an antigen of interest may also beprepared using traditional animal-based methods. These antigens, suchas, for example, peptides, can be conjugated at their N- or C-terminusto carrier proteins such as bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH) and used to immunize animals, such as rabbits, usingwell-known immunization regimes. Specific polyclonal antibodies can beobtained from the serum of the animal by, for example, affinitychromatography over a matrix containing the peptide used forimmunization bound to a solid support. Again, for example, antiseraraised against Tp17-like polypeptides can be adsorbed against otherpolypeptides bound to a solid support to remove any cross-reactiveantibodies, and vice-versa

Immunoassays

Any of a number of immunoassays may be used in the practice of themethods of the invention. For example, enzyme-linked immunosorbent assay(ELISA), agglutination assays, radioimmunoassays, turbidimetric assays,nephelometric assay, immunochromatography, chemiluminescence assays, andfluorescent assay. Such assays are well known in the art, and aredescribed in detail herein (Andreotti et al. (2003) Biotechniques35:850-859).

Methods for carrying out ELISA assays are well known in the art.Briefly, for detecting the presence of Tp17-like polypeptides in asample, for example, a solid phase, such as an ELISA plate, is coatedwith lysozyme. After washing, a sample that may contain a Tp17-likepolypeptide is added. The sample may be applied to several wells of theELISA plate, and detected via direct labeling (if appropriate), by usingan antibody to a Tp17-like polypeptide that is labeled, or by usingsecondary antibody and tertiary antibody or detection reagents(streptavidin-biotin) or labeled protein A or protein G. If the specificantibodies are differentially labeled, detection of more than oneantigen can occur in the same sample, for example in the same well ofthe ELISA plate. This requires use of labels that produce distinctsignals that can be independently quantified, for example, by using dyeswith different UV absorption maxima. Useful dyes and spectra include,but are not limited to, ABTS (2,2′-azinobis(3-ethylben-thiazoline-b-sulphonic acid) chromogenic substrate forhorseradish peroxidase (absorbs light at 410 nm) and TMB (3,3′,5,5′-Tetramethyl benzidine) chromogenic substrate for horseradishperoxidase (absorbs light at 450 nm after the addition of 1 M H₂SO₄.

Other known protein detection methods may be used in place of, or inaddition to the above immunoassays. For example, when using antibodiesagainst non-surface epitopes of proteins, enzyme or chemical (e.g.,CNBr) digestion of the proteins prior to detection may be used. Forexample, a full length Tp17-like polypeptide could be obtained using ageneric antibody that binds to a Tp17-like polypeptide, followed byenzymatic protein digestion and detection with Tp17-likepolypeptide-specific antibodies. Improved methods for CNBr digestion ofproteins are described in Kaiser et al. (1999) Anal. Biochem. 266:1-8.

Other methods that can be used include Western Blot, Far Western-Blot,immunohistochemistry, spot/slot blot techniques, protein chips, andbiosensors. For Western Blot, for example, duplicate protein samples maybe electrophoresed on an acrylamide gel and transferred to a membranesuch as nitrocellulose or PVDF. One blot is detected with antibody forTp17-like polypeptide and one blot is detected with antibody to acontrol protein. These primary antibodies are then detected, forexample, with labeled secondary antibodies. Alternatively, antibodiesspecific for a protein of interest and a control protein are eachlabeled with a different fluorescent dye and are reacted with the sameblot simultaneously. The fluorescence intensity of each dye is measured,and the ratio of the intensity indicates the ratio of the two proteins.

A Far Western blot involves the immobilization of a protein to a solidsupport, probing of the support with a ligand likely to bind directly toprotein and immunodetection of the bound protein-ligand. An exemplaryFar Western blot is shown in FIG. 5 and described in detail in Example2.

For immunohistochemistry, duplicate tissue sections may be treated withantibodies specific to a polypeptide of interest and a controlpolypeptide. These primary antibodies may be directly labeled or may bedetected with suitable secondary antibodies. Staining intensity can bemeasured with a charge-coupled device (CCD) camera and the proteinsquantitated. The ratio of the staining intensity indicates the ratio ofthe protein amounts. Alternatively, a single section can be stained withboth antibodies if the antibodies have been labeled with differentfluorescent labels.

Spot/slot blot techniques also are well known in the art. For example,identical amounts of a biological sample containing Tp17-likepolypeptide antibody, Tp17-like polypeptide-lysozyme complex antibody,or lysozyme antibody may be directly spotted onto a membrane anddetected with Tp17-like polypeptide, lysozyme, and/or the Tp17-likepolypeptide-lysozyme complex, as described above. The probes may belabeled or a secondary ligand may be used.

Many types of biosensor-based methods are known in the art and may beused for detecting and quantitating anti-Tp17-like polypeptideantibodies, anti-Tp17-like polypeptide-lysozyme complex antibodies, oranti-lysozyme antibodies. For example, samples containing antibodiesspecific to Tp17-like polypeptide and/or lysozyme may be bound to thesurface of the biosensor such that when Tp17-like polypeptide and/orlysozyme binds to the coated surface a detectable change occurs in someproperty of the surface. Biosensors measure, for example, mass changesat the surface, changes in electrical properties, or changes in opticalproperties. Each of these methods are well known in the art and aresuitable for use in the present methods.

Commercial biosensor-based methods are available from, for example,Biacore (Piscataway, N.J.) and are suitable for use in the presentinvention for detecting and quantitating changes in the levels ofantibodies to Tp17-like polypeptide and/or lysozyme. See also, forexample, the protein detection methods described in U.S. Pat. No.6,225,047, the contents of which are hereby incorporated by reference intheir entirety, and Davies et al. (1999) Biotechniques 27:1258-61.Commercial protein chip detection methods are available from Ciphergen(Fremont, Calif.). The invention can be scaled up to detect ordifferentiate between two or more pathogens, e.g., to determine if astrain of bacteria is a drug-resistant strain or is incapable of bindingto or inhibiting lysozyme.

In another embodiment, any of the methods described herein may becompetitive immunoassays, which are well known in the art. For example,a competitive sandwich immunoassay may be performed for measuring thelevel of antibody for a Tp17-like polypeptide and/or lysozyme in whichknown Tp17-like polypeptide antibodies are used to compete with theTp17-like polypeptide antibody in the sample for binding to Tp17-likepolypeptide. For example, serial dilutions of known Tp-like polypeptideantibodies may be incubated with the sample or with the Tp17-likepolypeptide prior to addition to the assay.

Mass spectrometric methods for protein detection may also be used todetect and quantify changes in the levels of antibodies, or fragmentsthereof, to Tp17-like polypeptide and/or lysozyme in a sample, forexample. See, for example, the methods described in U.S. Pat. Nos.5,719,060; 5,894,063; and Shimizu et al. (2002) J. Chromatogr. B.Analyt. Technol. Biomed. Life Sci. 25:776:15-30; Kiernan et al. (2002)Anal. Biochem. 301:49-56; and Pramanik et al. (2002) Protein Sci.11:2676-87. Mass spectrometry based protein detection methods are alsoavailable from Ciphergen (Fremont, Calif.).

Biological Sample

The sample analyzed or treated using any of the compositions and methodsof the invention may comprise a body sample such as blood, serum,plasma, tears, saliva, nasal fluid, sputum, ear fluid, genital fluid,breast fluid, colostrum, milk, placental fluid, perspirate, synovialfluid, ascites fluid, cerebrospinal fluid, bile, gastric fluid,gastrointestinal fluid, exudate, transudate, pleural fluid, pericardialfluid, semen, fecal material, upper airway fluid, peritoneal fluid,fluid harvested from a site of inflammation or other immune response,fluid harvested from a pooled collection site, bronchial lavage, urine,aqueous humor, biopsy material, material from the forestomach of aruminant animal, nucleated cell sample, fluid associated with a mucosalsurface, for example. Alternatively, the compositions and methodsaccording to the invention can be performed on dry cell samples (e.g.,hair or skin) or biopsy samples of any tissue in which the genes ofinterest or antibodies are expressed or deposited.

Other Samples

As referred to herein, “sample” also means any diagnostic, experimental,or clinical sample that may be suspected to contain lysozyme or aTp17-like polypeptide, or that otherwise requires testing or treatmentfor the presence of lysozyme or a Tp17-like polypeptide, or antibodythereto, such as a culture vessel, cuvette, swab, medical instrument(e.g., surgical instrument). A sample may also be a cell or fluidculture (e.g., throat aspirate, cerebrospinal fluid sample,hemoculture). A sample may be a pathogen preparation, such as, forexample, a bacteria, a virus, a parasite, a plasmid, a mycoplasma, amycotic agent (e.g., fungus, yeast), or prion preparation. The samplemay be a liquid or solid agent, such as a food, a medicine, an implant,a graft, a tissue or cell culture medium, a water sample, or othersolution, reagent or apparatus for which sterility is desired orrequired.

The compositions and methods can be used to diagnose or treat pathogencontamination or infection, e.g., pathogen-related diseases such as, forexample, syphilis, HIV infection, genital herpes, bubonic plague,dysentery, shigellosis, dental caries, E. coli infection, cysticfibrosis, tuberculosis, cholerae, group A and group B streptococcalinfections, staphylococcal infections, gastric ulcer, whooping cough,chlamydiosis, brucellosis, otitis media, meningitis, influenzainfection, malaria, salmonellosis, gonorrhea, vibriosis, colibacillosis,pneumonia, bronchitis, Severe Acute Respiratory Syndrome. In anembodiment, the compositions and methods of the invention are useful inthe diagnosis or treatment of cytoplasmic storage diseases such aslysosomal storage diseases.

In addition, the compositions and methods of the invention can be usedto diagnose or treat diseases such as cancer, infectious disease,inflammatory disease, Alzheimer's disease, renal amyloidosis, leukemia,Crohn's disease, and allergy.

Administration

Inhibitors of Tp17-like polypeptides that compete with Tp17-likepolypeptides for binding to lysozyme may be administered to a subject(e.g., a mammal, such as a human) in order to inhibit binding of aTp17-like polypeptides to the lysozyme, so that lysozyme is able tocombat the pathogenic agent. Alternatively, mutant lysozyme insensitiveto inhibition by Tp17-like polypeptides may be administered.

Any technical means (e.g., chemical drugs, competitor peptides,antibodies, vectors, siRNA) that interferes with the union of lysozymeand its cognate inhibitors (e.g., such as Tp17-like polypeptide) may beused prophylactically or therapeutically to combat pathogenic diseasecaused by any pathogen that comprises such an inhibitor (e.g., syphilis,HIV infection, genital herpes, bubonic plague, dysentery, shigellosis,dental caries, E. coli infection, cystic fibrosis, tuberculosis,cholerae, group A and group B streptococcal infections, staphylococcalinfections, gastric ulcer, whooping cough, Enterococcal infections,chlamydiosis, brucellosis, otitis media, meningitis, influenzainfection, malaria, salmonellosis, gonorrhea, vibriosis, colibacillosis,pneumonia, bronchitis, and Severe Acute Respiratory Syndrome, forexample. In an embodiment, the compositions and methods of the inventionare useful in the prophylaxis or treatment of cytoplasmic storagediseases such as lysosomal storage diseases.

Alternatively, ligands that compete with lysozyme for binding withTp17-like polypeptides may be administered to patients in order toinactivate lysozyme activity in patients that suffer from a diseaserelated to over production of lysozyme or any disease or condition inwhich a decrease in lysozyme activity is desired.

Preparations for oral administration of inhibitors of Tp17-likepolypeptide/lysozyme binding may be suitably formulated to givecontrolled release of the active compound. For buccal administration,the inhibitors may take the form of tablets or lozenges formulated in aconventional manner. Alternatively, an area may be swabbed, sprayed orapplied with inhibitors prior to obtaining a post-treatment sample(e.g., by scraping). For administration by inhalation, the inhibitorsfor use according to the methods of the invention is convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the inhibitors and a suitable powder basesuch as lactose or starch.

The inhibitors may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The inhibitors maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the inhibitors maybe in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use.

The inhibitors may also be formulated for rectal administration,inhibitors such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

The inhibitors may also be formulated as a depot preparation. Forexample, parenteral depot systems (PDS) are injected or implanted intothe muscle or subcutaneous tissue and incorporated drug released in acontrolled manner, allowing the adjustment of release rates overextended periods of time, ranging from several days up to one year. Suchlong acting formulations may be administered by implantation (e.g.,subcutaneously or intramuscularly) or by intramuscular injection. Theinhibitors may be formulated with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, such as a sparingly solublesalt. Other suitable delivery systems include microspheres which offerthe possibility of local noninvasive delivery of inhibitors over anextended period of time. This technology utilizes microspheres ofprecapillary size which can be injected via a coronary catheter into anyselected part of the body, e.g., the eye, or other organs withoutcausing inflammation or ischemia. The administered inhibitor is slowlyreleased from these microspheres and taken up by surrounding tissuecells.

Systemic administration of the inhibitors can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, e.g., for transmucosal administration bile salts and fusidicacid derivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution may be used locally to treatan injury or inflammation to accelerate healing.

Cells

The invention may require cells for the preparation or testing ofpathogen peptides, for example. Cultured primary or permanent animal orbacterial cell lines may be: primary cells (including, but notrestricted to, monocytes, synoviocytes, fibroblasts, and endothelialcells) derived from the same subject as the fluid sample or derived fromanother individual; permanent cell lines from a range of tissue andorgan origins (including, but not restricted to, cell lines availablefrom public access repositories such as American Tissue TypeCollection); or primary or permanent cell lines that have been stablytransfected with “promoter-readout” constructs.

Bacterial cell culture techniques are well known in the art. Forexample, bacterial cells may be grown in 2XYT broth on a shaker at 37°C. Methods for growing bacteria transformed with a plasmid or virus mayalso include growth in selective agents such as ampicillin or otherantibiotic. Preparations of cells and cell lysates, as well aspurification of proteins and nucleic acids are also well known in theart.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. Molecular Cloning—A LaboratoryManual (1989), 2^(nd) Ed., Sambrook et al. (Eds.) Cold Spring HarborLaboratory Press, Chapters 16 and 17; Hogan et al. (1986) Manipulatingthe Mouse Embryo: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,683,195; DNA Cloning,Volumes I and II (1985) Glover (Ed.); Oligonucleotide Synthesis (1984)Gait (Ed.); Nucleic Acid Hybridization (1984) Hames & Higgins (Eds.);Transcription and Translation (1984) Hames & Higgins (Eds.); Culture OfAnimal Cells (1987) Freshney, Alan R. Liss, Inc.; Immobilized Cells AndEnzymes (1986) IRL Press; Perbal (1984) A Practical Guide To MolecularCloning; Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (1987) Miller and Calos (Eds.) ColdSpring Harbor Laboratory; Methods In Enzymology, Vols. 154 and 155, Wuet al. (Eds.) Academic Press Inc., N.Y.; Immunochemical Methods In CellAnd Molecular Biology (1987) Mayer and Walker (Eds.) Academic Press,London; Handbook Of Experimental Immunology, Volumes I-IV (1986) Weirand Blackwell (Eds.).

The methods of the invention are useful therefore as a diagnostic and aprognostic tool, as a means for treating or immunizing against disease,for monitoring disease progression and resolution, for tracking theresponse, or lack thereof, to therapy, for evaluating the efficacy ofalternative or concomitant medication; and for establishing the correcttherapeutic dose of a medication. The methods of the invention may alsobe used, in the context of drug research and development, to assess thepotential efficacy and side effects of investigational and approveddrugs in biological samples collected in the course of animal testingand/or Phase I, II, III, and IV clinical trials and/or post-marketingstudies. ExamplesPractice of the invention will be more fully understoodfrom the following examples, which are presented herein for illustrativepurposes only, and should not be construed as limiting the invention inany way.

EXAMPLE 1 Chicken Lysozyme Copurifies with Recombinant Forms of theTreponema Pallidum 17 kDa Antigen

In the context of purifying E. coli-derived, recombinant T. pallidumTp17 antigen (GST-Tp17 or Tp17-HIS), a protein reproducibly co-purifiedwith Tp17. This “contaminant” protein, which had a molecular weight of14 KDa and did not react with an anti-Tp17 polyclonal serum, wasidentified as chicken lysozyme as described below.

A protein fraction containing both entities (Tp17 and the “contaminant”)was separated by PAGE-SDS, excised from the gel and subjected toN-terminal amino acid sequencing. The experiment yielded shortN-terminal peptide sequences (6-7 amino acids) that matched perfectlywith the expected sequence of the T. pallidum Tp17 antigen or chickenlysozyme, which was included in the cell paste resuspension buffer tofacilitate bacterial cell lysis. These observations suggested that Tp17interacts physically with chicken lysozyme. Experimental protocolscorresponding to the purification of GST-Tp17 and Tp17-HIS, as well asmicrosequencing experiments, are presented below.

GST-Tp17 Chromatography

The recombinant E. coli DH5α containing pGEX2T-Tp17 (Akins et al. (1993)Infect. Immun. 61:1202-1210) (FIG. 6) was grown in a 5 liter fermenterin 2×YT broth (Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual 2^(nd), Chris Nolan (Ed.) Cold Spring Harbor Press.) with 100μg/ml ampicillin (Roche Diagnostics, Barcelona, Spain). The culture wasinduced with 0.3 mM IPTG (Roche Diagnostics Mannheim, Germany) for fourhours. Cells were harvested and resuspended in 50 mM Tris (pH 8.0), 85mM NaCl, 2 mM EDTA, 1% Polyoxyethylene 10 tridecil ether, containingprotease a inhibitor cocktail (1 ml/g of cell paste, Sigma-Aldrich,Madrid, Spain) and 0.5 mg/ml chicken lysozyme (Sigma-Aldrich, Madrid,Spain). After incubation for 50 minutes, cells were sonicated andcentrifuged at 28,000 g. The recombinant GST-Tp17 was purified from thesupernatant by ion exchange chromatography (Q Sepharose XL, AmershamBiosciences, Cerdanyola, Spain). The flowthrough was further purified byglutathione affinity chromatography (glutathione sepharose FF, Amersham,Cerdanyola, Spain). A 100 mM reduced glutathione buffer was used toelute the GST-Tp17. The fractions collected were analysed by gelelectrophoresis. Another purification process was performed withoutusing lysozyme following the same protocol.

Tp17-HIS Chromatography

The gene sequence encoding the mature Tp17 protein coding sequence(including residues 23 to 156 of the Tp17 sequence deposited under theNCBI accession number P29722 (SEQ ID NO:31)) was PCR amplified using thepProExHT-Tp17 (Dr. Norgard, University of Dallas, Tex., USA) vector as atemplate. PCR amplification was performed using an Expand High FidelityPCR system kit (Roche Diagnostics, Mannheim, Germany) andoligonucleotides P17-NdeI (5′-AGA TAT ACA TAT GGT CTC GTG CAC AAC CGTGTG TCC GCA CGC CGG GAA GGC CAA-3′) (SEQ ID NO:33) and P17-rev1 (XhoI)(5′-ATG TAG CGA ACG GAG TTA-3′) (SEQ ID NO:34) under the conditionsrecommended by the supplier (Roche Diagnostics, Mannheim, Germany).Thermal cycling conditions were as follows: a denaturation cycle (1 min.at 94° C.) followed by 30 amplification cycles (1 min. at 94° C., 1 min.at 55° C., 1 min. at 72° C.). The resulting PCR amplicon (=300 bp) waspurified using the Nucleotrap purification system (Macherey-Nagel,Düren, Germany). 1 μg of purified PCR fragment and 1 μg of purifiedpET24a plasmid were each digested with 10 units of NdeI and 10 units ofXhoI using the conditions recommended by the supplier (RocheDiagnostics, Mannheim, Germany). Restricted fragments were separatedthrough a 0.8% agarose gel (Sambrook et al. (1989)) and fragmentscorresponding to Tp17 (≈300 bp) and pET24a (≈5300 bp) vectors wereexcised and gel purified using the Nucleotrap purification system(Macherey-Nagel, Düren, Germany). Both fragments were ligated using T4DNA ligase (Roche Diagnostics, Mannheim, Germany) using the conditionsrecommended by the supplier. The ligation reaction was then used totransform chemically competent E. coli TOP10 (Invitrogen SA, BarcelonaSpain) and transformants were selected on LB plates (Sambrook et al.(1989)) supplemented with 100 μg/ml of ampicillin (Roche Diagnostics,Mannheim, Germany). Plasmid DNA was prepared from 10 individual clonesand analyzed by restriction endonuclease digestion using NdeI and XhoI.Eight out of ten clones contained the correct pET24a-Tp17 recombinantplasmid. The sequence of one plasmid clone was verified by DNAsequencing (DNA Sequencing Facility, UAB, Barcelona, Spain) and wassubsequently used to transform the E. coli BL21 (DE3) Rosetta (Novagen,Madison, Wis.) expression strain. The resulting strain was named EcBK633(see FIG. 7).

The recombinant E. coli EcBK633 was grown in a 5 liter bioreactor in2×YT broth with ampicillin (Roche Diagnostics, Mannheim, Germany). Theculture was induced with 1 mM IPTG (Roche Diagnostics, Mannheim,Germany) for four hours. Cells were harvested and resuspended in 50 mMTris (pH 8.5) containing protease inhibitors (Sigma-Aldrich Madrid,Spain) and 0.6 mg/ml chicken lysozyme (Sigma-Aldrich, Madrid, Spain).After incubation for 45 minutes, cells were sonicated and centrifuged at30,100 g. The recombinant Tp17-HIS was purified from the supernatant bymetal ion affinity chromatography, using nickel-charged resin (ChelatingHP; Amersham, Cerdanyola, Spain) and a gradient elution 10-200 mMimidazole (Merck, Darhmstadt, Germany) and 500 mM imidazole asrecommended by the supplier. The fractions collected were analyzed bygel electrophoresis and Western Blot.

Electrophoresis and Western Blot Analysis

The chromatographic fractions and protein molecular weight markers (SeeBlue Plus III™, Invitrogen, Barcelona, Spain) were separatedelectrophoretically through two identical 15% SDS-PAGE gels as describedin Sambrook et al. (1989). Subsequently, one gel was stained withCoomasie Brilliant Blue R250 (Merck, Darhmstadt, Germany) in order todetect total protein. As shown in FIG. 8A, two major bands (14 kDa and17 kDa) were observed in the fraction corresponding to the Tp17 purifiedprotein. The proteins in the other gel were electro-transferred to anImmobilon™ P PVDF membrane (Millipore Corp., Bedford, Mass.) asrecommended by the supplier. The membrane was then incubated for 1 hourat room temperature (18-22° C.) in 5 ml of Blotto™ (BioRad, Hercules,Calif.) containing PBS, 0.05% w/v Tween and 10% w/v of dried, non-fatskimmed milk (PBST). The membrane was then processed as follows: (1)three 10 minute washes with PBST, (2) 1 hour incubation at roomtemperature (18-22° C.) in the presence of 10 mls of a human serum froma patient with syphilis diluted 1/200 in Blotto™, (3) three 10 minutewashes with PBST, (4) 1 hour incubation at room temperature with 10 mlsan alkaline phosphatase-conjugate, rabbit-anti-human polyclonal serum(BioRad, Hercules, Calif., USA) diluted 1/2000 in PBST, (5) three 10minute washes with PBST, and (6) a final incubation with 5 ml ofNBT/BCIP alkaline phosphatase chromogenic substrate (Sigma-Aldrich,Madrid, Spain) until color development As shown in FIG. 8B, this assaydetects binding of the human IgG to the 17 kDa (Tp17-HIS) but not to the14 kDa protein.

N-Terminal Sequencing

The chromatographic fraction containing the purified Tp17-HIS with thecontaminant protein (14 kDa) and a protein molecular weight marker(SeeBlue Plus II™, Invitrogen SA, Barcelona, Spain) was separatedelectrophoretically through a 15% SDS-PAGE gel as described in Sambrooket al. (1989). Subsequently, the proteins in the gel wereelectro-transferred to an Immobilon™ P PVDF membrane (Millipore Corp.,Bedford, Mass.) using the suppliers guidelines. The membrane was stainedwith Coomassie Brilliant Blue R250 and the two bands were cut and driedfor sequencing analysis. The N-terminal analysis by automatic Edmandegradation was performed in a Beckman LF3000 sequencer with a PTH-aminoacid analyzer (System Gold, Beckman Coulter, Fullerton, Calif.).

The sequences obtained were: _(NH2-)VS(C)TTV_(-COOH) for the 17 KDaprotein (SEQ ID NO: 35). _(NH2-)KVFGR(C)ELAA_(-COOH) for the 14 KDaprotein (SEQ ID NO: 36).

As shown in FIG. 8B, the 17 kDa protein N-terminal sequence correspondedto the Treponema pallidum Tp17 and the 14 kDa protein sequencecorresponded to the chicken lysozyme.

EXAMPLE 2 Far Western Detection of Tp17 Binding to Chicken and HumanLysozyme

The observation that the T. pallidum 17 kDa (Tp17) protein antigencopurifies with chicken lysozyme strongly suggested a direct physicalinteraction between both proteins. In order to test this hypothesis, aFar-Western blot lysozyme-protein interaction assay was performed. Theassay is summarized in FIG. 5 and comprises generally the followingthree steps: (1) immobilization of purified lysozyme onto a membrane;(2) probing of the membrane with a ligand likely to bind directly to thelysozyme; and (3) immunodetection of the bound lysozyme-ligand. Adetailed experimental procedure is presented below.

Far Western Blotting Detection of Lysozyme-Tp17 Protein Interactions

Purified chicken egg white lysozyme and human breast milk lysozyme werepurchased from Sigma-Aldrich (Madrid, Spain). Two series of aliquotfractions containing 1 μg, 5 μg, and 10 μg of purified lysozyme and aprotein molecular weight marker (SeeBlue Plus II™, Invitrogen SA,Barcelona, Spain) were separated electrophoretically through a 4-12%SDS-PAGE gel using the conditions recommended by the supplier(Invitrogen). The gel was then processed for Western blotting asdescribed in Sambrook et al. (1989) according to art known methods, andthe proteins were electro-transferred to an Immobilon™ P PVDF membrane(Millipore Corp., Bedford, Mass.). The presence of lysozyme immobilizedonto the PVDF membrane was assessed by staining for 1 minute withPonceau S stain (Sigma-Aldrich, Madrid, Spain), and then destained byvigorous washing for 5 minutes with desionized water. The membrane wasdivided in two, each portion containing a protein ladder and threequantities of chicken or human lysozyme (1 μg, 5 μg and 10 μg per lane).Each membrane was then incubated for 16 hours at 4° C. in 5 ml of TBST(10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% weight/vol. Tween 20™,)supplemented with 5% (weight/vol.) of non-fat skimmed driedSVELTESSE™milk (NESTLÉ, ESPAÑA, Barcelona, Spain) (to make TBST-milk).The membranes were then processed separately as follows: (1) three 1minute washes with 10 mls TBST, (2) 1 hour incubation at roomtemperature (18-22° C.) in the presence of 10 mls of 100 mg/ml GST-Tp17fusion protein (Akin et al. (1993)) or a control protein GST-Tp47, (Hsuet al. (1989) Infect. Immun. 57:196-203; Weigel et al. (1992) Infect.Immun. 60:1568-1576) (3) three 10 minute washes with 5 mls of TBST, (4)30 minute incubation at room temperature with 10 mls of an anti-GSTpolyclonal goat serum (Amersham Biosciences, Cerdanyola, Spain) diluted1/7500 with TBST-milk, (5) three 10 minute washes with 5 ml of TBST atroom temperature, (6) 1 hour incubation at room temperature with 10 mlsof an alkaline phosphatase-conjugate, rabbit-anti-goat polyclonal serum(Dako A/S, Glostrup, Denmark) diluted 1/5000 in TBST-milk, (7) three 10minute washes with 5 ml of TBST and (8) 3 minutes incubation with 5 mlof Nitroblue tetra zolium/Bromo Chloro Indolyl Phosphate alkalinephosphatase chromogenic substrate (NBT/BCIP) (Sigma-Aldrich, Madrid,Spain) until color development.

The above assay detected binding of the GST-Tp17 fusion protein to bothchicken and human lysozyme (14 kDa band in FIGS. 9B and 9D,respectively). Moreover, the binding of GST-Tp17 to lysozyme is strongsince it resisted numerous washings in the presence of detergent (0.05%Tween 20™. No signal was detected when the membrane was incubated withcontrol GST-Tp47 antigen (FIGS. 9A and 9C), demonstrating that theinteraction between Tp17 and lysozyme is specific and does not involvethe GST moiety, which is shared by both GST-Tp17 and GST-Tp47. Theseresults demonstrate that (i) the T. pallidum Tp17 antigen binds stronglyand specifically to human and chicken lysozyme, and (ii) this propertyis contained between residues 22 to 156 of Tp17 (numbers relate to theTp17 peptide sequence deposited under accession number P29722 (SEQ IDNO:31)). This assay is used to characterize novel lysozyme bindingproteins as well as to identify lysozyme mutants unable to bindlysozyme's cognate inhibitor. Conversely, this assay is used to screenfor Tp17 mutants unable to bind or associate with lysozyme. This assayis also used to screen for substances (e.g., peptides, proteins, drugs,antibodies, nucleic acids, PNAs, etc.) that interfere with the bindingof lysozyme to its cognate inhibitor.

EXAMPLE 3 The T. Pallidum Tp17 Protein Antigen Inhibits theAntibacterial Activity of Chicken and Human Lysozyme

Lysozymes are well characterized antibacterial agents found on mucosalsurfaces and in biological fluids. Due to their potent acetyl-muramidaseenzymatic activity, lysozymes are capable of hydrolyzing cell wallpeptidoglycan, thereby killing many pathogenic bacteria. The strongbinding between Tp17 and human lysozyme suggested that this binding mayalter the antibacterial activity of lysozyme. This hypothesis isconsistent with the observation that (i) T. pallidum is a mucosalpathogen and (ii) it is in contact with human lysozyme in its ecologicalniche, throughout its infectious life cycle.

To test this hypothesis, the antibacterial activity of both human andchicken lysozyme, in the presence or absence of GST-Tp17, was assayedusing an EnzCheck® lysozyme assay kit (Molecular Probes, Eugene, Oreg.).The assay comprises the use of Fluorescein-labeled Micrococcuslysodeikticus bacterial cells as a fluorescent substrate of lysozyme. Inthis Gram positive bacterium, the peptidoglycan layer is directlyaccessible to lysozyme, and has been Fluorescein-labeled such thatfluorescence is naturally quenched. Upon lysozyme hydrolysis, quenchingis relieved and fluorescein is released in a quantity that isproportional to lysozyme activity. The following experimental protocolwas used.

Lysozyme Inhibition Assay

A typical 100 μl reaction consisted of 25 μl of lysozyme solution (10units for chicken lysozyme or 50 units for human lysozyme, dissolved indeionized water), 25 μl of the test protein solution and 50 μl of asuspension of fluorescein-labeled Micrococcus lysodeikticus (50 μg/ml).The reaction was incubated for 45 minutes at 37° C. and the fluorescencewas measured (λ_(exc)=485 nm; λ_(emi.)=520 nm) using a fluorescencemulti-well plate reader model FLx800 (Bio TEK instruments, Winoosky,Vt.) equipped with KC junior data acquisition software (Bio TEKInstruments, Winoosky, Vt.). Each experimental point was expressed asthe mean and standard deviation corresponding to the reading of threeindependent wells. Wells containing 50 μl of deoinized water and 50 μlof Fluorescein-labeled Micrococcus lysodeikticus suspension were used asa blank. Two fold serial dilutions of test protein were used to vary theconcentration of the test protein from 83.5 to 1.3 μg/well.

As shown in FIG. 10, the acetyl muramidase activity of both human andchicken lysozyme was strongly inhibited (>85% of inhibition) by theaddition of as little as 1.3 μg/well of the GST-Tp17 fusion protein. Incontrast, the control GST-Tp47 fusion protein did not exert anyinhibitory effect on either human or chicken lysozyme's acetylmuramidase enzymatic activity. These data demonstrate that Tp17specifically inhibits the antibacterial activity of both chicken andhuman lysozymeand does not depend upon the GST moiety. Additionally,this assay is used to characterize novel lysozyme inhibitors as well asto identify lysozyme mutants unable to bind its cognate inhibitor and/orthat is insensitive to inhibition by Tp17, Ivy, or other Tp17-likeproteins. The assay also provides a useful screen for substances (e.g.,peptides, proteins, drugs, antibodies, nucleic acids, or PNAs, etc.)that interfere with the inhibition of lysozyme's antibacterial activity.

EXAMPLE 4 Structural Determinant Required for Binding of EscherichiaColi Ivy to Chicken Lysozyme

In order to identify the polypeptide region(s) involved in the bindingof T. pallidum Tp17 to lysozyme and the inhibition of lysozyme acetylmuramidase activity, biological databases were searched for polypeptidespreviously described to bind to and inhibit the acetyl muramidaseactivity of lysozyme. A search through the National Institute ofHealth's PubMed database, using the terms “lysozyme” and “inhibitor” askey words, identified an Escherichia coli protein named Ivy (standingfor inhibitor of vertebrate lysozyme), reported to bind and inhibit theacetyl muramidase activity of both human and chicken lysozyme (Monchoiset al. (2001) J. Biol. Chem. 276:18437-18441)). This protein was ofinterest since (i) it is similar in size (14 kDa mature polypeptide) tomature Tp17 (15 kDa mature polypeptide) and (ii) a crystal structure ofIvy complexed to chicken lysozyme was available in the protein data bankPDB at the Research Collaboration for Structural Bioinformatics (RCSB)under the accession number 1GPQ. However, as represented in FIG. 11A,the Ivy protein shares less than 21% of amino acid sequence identitywith Tp17 and no obvious peptide motif could be deduced from polypeptidesequence alignments. The Ivy/chicken lysozyme three dimensional crystalstructure was examined for residues located at the interface betweenboth molecules. The Cn3D three dimensional molecular structure viewerwas used to analyze the molecular interactions between Ivy and chickenlysozyme. In the following examples, all the numbering refers andcorresponds to the data archived under the following NCBI accessionnumbers: P00698 for chicken lysozyme (SEQ ID NO:311), NP_(—)000230 forhuman lysozyme (SEQ ID NO:302), P45502 for E. coli Ivy (SEQ ID NO:312),and P29722 for T. pallidum Tp17 (SEQ ID NO:31).

As depicted in FIG. 1I B, three zones of possible contact of chickenlysozyme with Ivy were identified. The corresponding peptide stretchesare:

Peptide II displayed a finger-like structure composed of a cys-cysdisulfide bridge closing at both ends a peptide loop comprising a KPHDmotif (SEQ ID NO:40; FIG. 14A). A refined analysis of the amino acidpresent in this loop revealed two strong non-covalent, electrostaticinteractions: between H_(88-ivy) and E_(53-chkcLys), and betweenD_(89-ivy) and R_(132-chkcLys).

The nitrogen atom of H_(88-ivy) interacted with the oxygen atom ofE_(53-chkcLys) and the oxygen atom of D_(89-ivy) interacted with thenitrogen atom of R_(132-chkcLy) (FIG. 12). Using the 3-D Mol viewercomputer software (Vector NTI suite 8.0, Informax, Frederick, Md.), thedistance between atomic centers was measured at 2.56 Å (1 angstrom=10⁻¹⁰meter) in the H_(88-ivy)/E_(53-chkcLys) interaction and 2.46 Å in theD_(88-ivy)/R_(132-chkcLys) interaction. The nature (electrostaticattraction between lateral chain residues) and the proximity (in therange 2.4-3.4 Å) of atomic centers indicated the presence of aparticular class of non-covalent bond referred to as a “salt bridge.”This observation was interesting because most salt bridges occur inrelatively water-free depressions or at biomolecular interfaces wherewater is excluded. (Petsko et al. (2003) Protein Structure and Function.Lawrence and Robertson (Eds.) New Science Press Ltd., UK).

FIGS. 13A-13D illustrate that the salt bridge involving D_(89-ivy)

R_(132-chkcLys) was partially exposed to the water molecules of thesolvent (FIGS. 13C and 13D), while the salt bridge involving H_(88-ivy)

E_(53-chkcLys) was completely buried and consequently protected from thesolvent by the protein-protein interface (FIGS. 13A and 13B). Given thathydration of a salt bridge reduces its bonding strength, the H_(88-ivy)

E_(53-chkcLys) salt bridge is less likely to be broken by the solventthan the D_(89-ivy)

R_(132-chkcLys) salt bridge. Interestingly, FIGS. 13A and 13B also showthat the H_(88-ivy)

E_(53-chkcLys) salt bridge blocked the accessibility to the glutamicacid 53 (E₅₃) of chicken lysozyme. Glutamic Acid number 53 (E₅₃) andAspartic acid 71 (D₇₁) in the full length lysozyme sequence correspondto residues E₃₅ and D₅₃ of the mature lysozyme peptide chain. Asdepicted in FIGS. 3A and 3B, E₅₃ together with D₅₃ in mature chickenlysozyme (or in human lysozyme) are conserved across all known lysozymesequences and are thought to be the two catalytic residues required forthe hydrolysis of the peptidoglycan (Malcolm et al. (1989) Proc. Natl.Acad. Sci. USA 86:133-7).

Taken together, these data demonstrate that Ivy establishes two saltbridges with lysozyme: one essential for the specificity and thestability of the interaction (H_(88-ivy)

E_(53-chkcLys)) as well as for the inhibition of lysozyme antibacterialactivity by steric hindrance, and the other (D_(89-ivy)

R_(132-chkcLys)) involved to a lesser extent in stabilizing thelysozyme/inhibitor interaction. Due to the strict conservation ofresidues E₃₅ and D₅₃/D₅₂ D₅₂ (numbering refers to the mature lysozymesequence) across species, these observations demonstrate that Tp17inhibits a wide range, if not all, lysozymes.

EXAMPLE 5 Identification of a Consensus Peptide Sequence Shared byProtein Inhibitors of Mammalian Lysozymes

Example 4 demonstrated that the peptide II sequence_(NH2)-CKPHDCG-_(COOH) (SEQ ID NO:38) is necessary for binding to andinhibiting the enzymatic activity of chicken lysozyme. A detailedexamination of the Tp17 polypeptide sequence identified two candidatesequences possibly mediating binding and inhibition of both chicken andhuman lysozyme. Both sequences, referred to as Tp17_pep1 and Tp17_pep2,are listed below:

-   Tp17_pep1: _(NH2-29)C P H A G K A K A E K V E C_(42-COOH) (SEQ IDS    NO:41)-   Tp17_pep2: _(NH2-114)K A P H E K E_(120-COOH) (SEQ ID NO:42)

Tp17_pep1 is closely related to the _(NH2-85)CKPHDCG_(91-COOH) motif(SEQ ID NO:38) of E. coli Ivy protein but differs by the presence of anine amino acid stretch separating the histidine residue from theaspartic acid residue. As shown in FIG. 14B, and as found for the Ivymotif (FIG. 14A), bridging of the two flanking cysteines likely bringsthe critical histidine and aspartic acid residues in close spatialproximity, possibly in a configuration able to contact with residues E₅₃and R₁₃₂ of lysozyme. Although the function of the possible 9 amino acidloop of Tp17 is presently unknown, its composition suggest that it ishighly hydrophilic and probably immunogenic. Positively charged lysineresidues contained in this 9 amino acid loop may contact directly with anegatively charged surface area of lysozyme, providing as well anadditional anchorage point to strengthen the binding. By aligning theTp17_pep1 peptide with the Ivy lysozyme binding motif, the consensussequence CX₁PHX_(n) X₂C (SEQ ID NO:43), wherein X₁ is at least one aminoacid, is absent, or a peptide bond, X_(n) is from zero to nine aminoacids, or a peptide bond, and X₂ is glutamic acid or aspartic acid, wasobtained.

Although lacking the two flanking cysteines, the Tp17_pep2 is alsoclosely related to the _(NH2)-CKPHDCG-_(COOH) motif SEQ ID NO:38 of theE. coli Ivy protein, since it shares the critical PH residues followedby an acid residue (D/E). Compiling structural data, bibliographicinformation and databases searches revealed a lysozyme binding consensus[CKVA][ACK]P H [AED][CGK](SEQ ID NO:3), written using the universalPROSITE peptide pattern syntax format.

Additional consensus sequences were determined and were written usingthe universal PROSITE peptide pattern syntax format, which is availablethrough the Expert Protein Analysis System's SWISSProt database, asfollows:

(a) X_(n)PHX_(n) (SEQ ID NO:1), wherein X_(n) is at least one aminoacid.

(b) CX₁X₂X₃PHX₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃C (SEQ ID NO:2), wherein X₁ to X₁₃are any amino acid, no amino acid or a peptide bond.

(c) X₁CPHAG (SEQ ID NO:25), wherein X₁ is C or V.

Preferred embodiments of Tp17-like polypeptides are illustrated in FIGS.1A, 1B, and 2.

EXAMPLE 6 In Silico Identification of Putative Lysozyme Binding Proteins& Inhibitors

Given the low overall peptide sequence conservation shared by E. coliIvy and T. pallidum Tp17, previously undetected lysozyme binding motifswere likely present in the genome of other pathogenic organisms. To testthis hypothesis, the consensus sequences defined in Example 5 were usedto scan protein databases (Swiss-Prot, TrEMBL, TrEMBLnew and PDB) usingthe ScanProsite algorithm (http://us.expasy.org/tools/scanprosite/).FIGS. 1A, 1B, and 2 illustrate that proteins containing a peptidestretch fitting within the consensus sequences were present in numeroushuman and animal pathogens.

Proteins from T. denticola, B. thetaiotaomicron, C. burnetti, H.influenzae, N. meningitidis serogroup A and serogroup B, V. cholerae, Vvulnificus, H. ducreyi, S. typhi, L. pneumophila, S. aureus, N.gonorrhoeae, and B. pertussis, can be grouped into the followingconsensus sequence: CX(0,3)PHX(0,14)C, which corresponds to Cys Xaa₁Xaa₂ Xaa₃ Pro His Xaa₄ Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa₁₇ Cys (SEQ ID NO:176), wherein Xaa is any aminoacid or is absent.

Proteins (from P. gingivalis and H. pylori) are slightly atypicalvariants that could be gouped under the following consensus:CX(0,3)HX(0,10)C which corresponds to Cys Xaa₁ Xaa₂ Xaa₃ His Xaa₄ Xaa₅Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys (SEQ ID NO: 175),wherein x is any amino acid or is absent.

This result is interesting since all of these organisms are mucosalpathogens during part or most of their infectious life cycle. Ofparticular interest was the presence of a consensus peptide motif in thecarboxy terminus of the Herpes Simplex virus type 2 gJ glycoprotein.Although the function of this protein is presently unknown, it isencoded by the Us5 gene present in the unique short (Us) region of thevirus. This region is known to contain most of the HSV proteins involvedin virulence and mediating protein-protein interactions with human hostproteins, supporting a role of gJ in the pathogenesis of HSV infection.Interestingly, several publications have documented that lysozyme has astrong inhibitory effect upon HSV2 infection and its clinicalmanifestations (Cisani et al. (1989) Microbios. 59:73-83; Oevermann etal. (2003) Antivir. Res. 59:23-33). The gJ protein from HSV-2 is also alysozyme binding/inhibitor protein. Strikingly, the consensus lysozymebinding motif is absent from the closely related HSV-1 gJ protein (FIG.15). This observation provides a possible explanation for clinicalobservations documenting a preferential but not exclusive tropism ofHSV-1 for the orofacial region whereas HSV-2 is found most frequently inthe lysozyme-rich, genital mucosa (Lowhagen et al. (2002) Acta Derm.Venereol. 82:118-21; Bruisten et al. (2003) Curr. Womens Health Report3:288-98). In addition, it is intriguing to observe that two genitalpathogens, one virus (HSV-2) and one bacteria (T. pallidum), share acommon lysozyme inhibitory peptide sequence.

These observations demonstrate that gJ, alone or combined with humanlysozyme, is useful for discriminating between HSV-1 and HSV-2infections using differential serological or virological diagnosticassays. It also provides compositions and methods for treating Herpessimplex infections with lysozyme mutants that can escape inhibition bygJ and/or members of the Ivy and Tp17 families.

EXAMPLE 7 Identification of Point Mutations Destabilizing theLysozyme/Inhibitor Protein-Protein Interaction

It is widely recognized that protein-protein interactions depend uponstructural and physicochemical complementarity. The irregularity andlocal heterogeneity of protein surfaces enables specific binding andassociation with ligands and provides a basis for the formation ofquaternary structure. The fit between one protein surface and anotherdepends upon more than their shape. It extends to the weakphysicochemical bonds that hold complexes together: covalent bonding,hydrogen bonding, salt bridges, long range electrostatic interactionsand Van der Waals interactions. This property of complementarity isuniversal, whether between a protein and a small molecule or between aprotein and another kind of macromolecule (Petsko et al. (2003)).

Mutations localized in the lysozyme coding sequence, selected so thatacetyl muramidase and antimicrobial activities are not affected,destabilize the lysozymes/inhibitor interaction and confer resistance toinhibition by pathogen proteins such as the T. pallidum Tp17 antigen ormembers of the Ivy family. In an embodiment, the mutant lysozymesubstantially retains the desired anti-microbial activity (e.g., atleast about 30%, 50%, or even 80%) of the corresponding wild-typelysozyme. Such mutant lysozymes represent novel and interesting toolsfor the treatment of infectious diseases. They are also substitutes fornatural hen egg-derived chicken lysozyme used in commercially availablemedicinal preparations and have improved potency and spectrum ofactivity. A list of potential drugs likely to be improved by the use ofsuch inhibition-resistant lysozyme mutants is given below: Marketed DrugPharmaceutical Company LYSOPAINE ORL Boehringer Ingelheim LYSO-6 UCBHealth Care GLOSSITHIASSE Laboratoire Jolly-Jatel CANTALENE LaboratoireCooper HEXALYSE Laboratoire Bouchara-Recordati

Such lysozyme mutants are obtained using both targeted and randomapproaches.

Targeted Identification of Inhibition-Resistant Lysozyme Mutants Deducedfrom Crystallographic Data

Due to their elevated isoelectric point (pI), human lysozyme(theoritical pI=9.28) and chicken lysozyme (theoritical pI=9.32) exhibita strong positive charge at physiological values of pH comprised betweenpH 3 and pH 7. All known lysozyme inhibitors have low pI values. Forexample, mature Ivy proteins (Monchois et al. (2001) J. Biol. Chem.276:18437-18441) from E. coli (pI=5.51) and P. aeruginosa (pI=5.69), aswell as the mature Sic protein protein (Fernie-King et al. (2002)Infect. Immun. 70:4908-4916) from S. pneumoniae (pI=4.39) are negativelycharged at physiological pH. It was hypothesized that positively chargedlysozyme residues that are present in the lysozyme/inhibitor contactarea are attractive targets for mutagenesis. Producing a lysozyme mutanthaving a negatively charged contact area might produce an electrostraticrepulsion preventing binding of the inhibitor. Since these mutations donot affect lysozyme catalytic residues, they are likely to produceactive lysozyme mutants, uninhibited by pathogens.

The E. coli Ivy/chicken lysozyme crystal structure deposited under thePDB accession number 1GPQ, as well as the P. aeruginosa Ivy crystalstructure deposited under the PDB accession number 1HKE, were inspectedwith a VNTI 3D molecular viewer. Critical residues were identified oneach lysozyme and the following point mutations altering the binding ofthe inhibitor were made: Position in Position in Human Lysozyme ChickenLysozyme (NP_000230 (P00698 (SEQ ID NO: 302)) (SEQ ID NO: 311)) Mutatedto: Mutation affecting lysozyme surface charge Lys₁₉ Lys₁₉ Asp, Glu, orGly Arg₂₃ Arg₂₃ Asp, Glu, or Gly Lys₅₁ Lys₅₁ Asp, Glu, or Gly Arg₁₃₁Arg₁₃₀ Asp, Glu, or Gly Arg₁₃₃ Arg₁₃₂ Asp, Glu, or Gly Mutation steri-cally hindering binding of the inhibitor Gly₅₅ Asn₅₅ Phe, Trp or TyrAsn₅₇ Asn₅₇ Phe, Trp or Tyr Asn₁₃₂ Asn₁₃₁ Phe, Trp or Tyr

Introduction of Point Mutations by Site-Directed Mutagenesis:

Mutations are introduced using the QuikChange® Site-Directed MutagenesisKit, following the guidelines recommended by the supplier (Stratagene,La Jolla, Calif.). Briefly, the QuikChange® Site-Directed MutagenesisKit is used to make point mutations, switch amino acids, and delete orinsert single or multiple amino acids. The QuikChange® site-directedmutagenesis method is performed using PfuTurbo® DNA polymerase and atemperature cycler. PfuTurbo® DNA polymerase replicates both plasmidstrands with high fidelity and without displacing the mutantoligonucleotide primers. The basic procedure utilizes a supercoileddouble-stranded DNA (dsDNA) vector with an insert of interest and twosynthetic oligonucleotide primers containing the desired mutation. Theoligonucleotide primers, each complementary to opposite strands of thevector, are extended during temperature cycling by PfuTurbo® DNApolymerase. Incorporation of the oligonucleotide primers generates amutated plasmid containing staggered nicks. Following temperaturecycling, the product is treated with Dpn I. The Dpn I endonuclease(target sequence: 5′-Gm 6 ATC-3′) is specific for methylated andhemimethylated DNA and is used to digest the parental DNA template andto select for mutation-containing synthesized DNA. DNA isolated fromalmost all E. coli strains is dam methylated and therefore susceptibleto Dpn I digestion. The nicked vector DNA containing the desiredmutations is then transformed into XL1-Blue supercompetent cells. Thesmall amount of starting DNA template required to perform this method,the high fidelity of the PfuTurbo DNA polymerase, and the low number ofthermal cycles all contribute to the high mutation efficiency anddecreased potential for generating random mutations during the reaction.

Random Isolation of Inhibition-Resistant Lysozyme Mutants UsingPCR-Mediated Random Mutagenesis Followed by a Microbial Two-HybridScreening

The two-hybrid system is based on the observation that many eukaryotictranscriptional activators consists of two physically discrete modulardomains: the DNA-binding domain (DNA-BD) and the activation domain (AD).The DNA-BD binds to a specific promoter sequence and the AD directs theRNA polymerase II complex to transcribe the downstream gene conferring aselectable/screenable phenotype. The domains act as independent modules:neither alone can activate transcription, but each domain continues tofunction when fused to other proteins. Suitable commercial systemsinclude the ready-to-use BacterioMatch® II Two-Hybrid System(Stratagene, La Jolla, Calif.) or the BD Matchmaker™ system (BDBiosciences Clontech, Palo Alto, Calif.,.

Lysozyme and Tp17, one fused to the AD and the other fused to DNA-BD,are expressed. If the two proteins interact, the DNA-BD and the ADdomains are brought into close proximity and activate transcription of areporter gene. Depending upon the host system used (e.g., bacterial,yeast or mammalian), interaction is detected by screening for observablephysiological or structural change (e.g., color of the colony, emissionof fluorescence, enzymatic activity, etc.). For example, AD and DNA-BDdomains are fused with lysozyme and Tp17 respectively, transformed intoan appropriate microbial host (e.g., bacteria or yeast), and theresulting transformants are screened for the appearance of blue coloniesindicative of the activation of the reporter gene (e.g.,beta-galactosidase). Once clones are obtained, plasmid DNA sequencebearing the human lysozyme open reading frame is subjected to extensivePCR-mediated random mutagenesis. The resulting transformants arevisually screened for mutants that have lost the blue color and arelikely to bear one or more mutations that impair the lysozyme/Tp17-likepolypeptide interaction. Plasmid DNA from the corresponding clones isthen extracted and sequenced.

EXAMPLE 8 Human But not Chicken Lysozyme Improves the Sensitivity ofHemagglutination Kits Used For Syphilis Diagnostics

Example 3 demonstrates that, in vitro, the T. pallidum 17 kDa proteinTp17 strongly binds to chicken and human lysozyme, and strongly inhibitsthe antibacterial activity of both lysozymes. The following experimentdemonstrates that these binding and inhibition interactions also occurin vivo during the infection of human mucosal surfaces by T. pallidum.As a consequence of Tp17 binding to lysozyme, new epitopes are likelyformed and exposed to the immune system. Syphilitic patients may raisean immune response against Tp17 epitopes as well as against epitopesshared by the Tp17-lysozyme complex or even lysozyme alone, as shown inFIG. 4. To test this, human or chicken lysozyme was added to thehemagglutination reagents used for a syphilis diagnostic assay in orderto detect those antibodies generated against the complex, therebyimproving the sensitivity of the assay. An increase in sensitivity ofthe syphilis assay was observed that was specific for human lysozyme.

Purified chicken egg white lysozyme (L-6876) and human breast milklysozyme (L-6394) were obtained from Sigma-Aldrich (Madrid, Spain). Anamount of chicken or human lysozyme sufficient to maintain theproportion: 3 μg lysozyme/1 μg Tp17 was added to a hemagglutinationreagent (Biokit SA, Lliça d'Amunt, Spain) prepared as follows. Chickenred cells were tanned, fixed, and coated with GST-Tp17 (40 μg/ml) aspreviously described (Herbert et al. (1979) Passive HaemagglutinationWith Special Reference to the Tanned Cell Technique. Handbook ofExperimental Immunology (vol. 1), pgs 20.1-22.20 (DMWeir (Ed.) BlackwellScientific Publications (Oxford)); Stefen et al. (1951) J. Exp. Med.93:107-120; Hirata (1968) J. Immunol. 100:641-46). One batch of GST-Tp17coated, fixed chicken red cells (5 ml), was supplemented with 6 μl ofpurified chicken egg white lysozyme (100 μg/111). Another batch ofGST-Tp17 coated, fixed chicken red cells (1 ml), was supplemented with120 μl of human breast milk lysozyme (1 μg/μl). Both lots of sensitizedred cells were homogenized by magnetic stirring, for 30 minutes at roomtemperature. The sensitivity of each lot was then evaluated byperforming a hemagglutination reaction using a set of human syphilissera, characterized to give a borderline response with other commercialhemagglutination reagents.

The hemagglutination reaction was performed as follows. In around-bottom microtiter plate (U-shaped), 25 μl of diluent (SyphagenTPHA, Biokit SA, Barcelona, Spain) was added to well 1, 100 μl to well 2and 25 μl to each of wells 3 to 8. 25 μl of sample was added to well 1.The content of well 1 was mixed and 25 μl was transferred to well 2 andmixed. 25 μl from well 2 to well 3. Serial two-fold dilutions were thenmade through well 8. 25 μl from well 8 was discarded. 75 μl ofhemagglutination reagent was added to wells 3-8 (the final sampledilutions were 1/80 to 1/2560). The contents of the wells were mixedusing a shaker for at least 30 seconds. The plate was covered andincubated for 1 hour at room temperature. The results of the assay wereread applying the following criteria A result was scored positive eitherwhen a smooth mat of cells covering the entire bottom of the well orwhen a smooth mat of cells partially covering the bottom of the well andsurrounded by a heavy ring of cells was observed. A result was scorednegative either when a button of cells with a small central opening orwhen a button of cells with or without a very small hole in the centerwas observed. The titer of a sample corresponded to the highest dilutiongiving a positive reaction. The cut-off of this technique was 1/80.

As shown in FIGS. 16A and 16B, the addition of human lysozyme to theassay improves the intensity of hemagglutination (i.e., hemagglutinationoccurred at higher dilutions in the presence of human lysozyme comparedto the absence of lysozyme) in a concentration dependent manner. Thisimprovement appeared to be specific for human lysozyme because theaddition of chicken lysozyme did not enhance the response. The additionof human lysozyme drastically improved the sensitivity of the syphilishemagglutination reagent and allowed for the detection of antibodiesthat are not detected in kits presently in the market.

EXAMPLE 9 Both Natural and Recombinant Human Lysozyme Improve theSensitivity of Hemagglutination Test Used for Syphilis Diagnostics

Human breast milk is a natural source of human lysozyme. However,purifying lysozyme from this body fluid is tedious due to: (i) its lowabundance, (ii) the difficulty in ensuring a regular and reproduciblesupply, (iii) the possible contamination with undetected pathogen, aswell as (iv) social and ethical concerns. A recombinant source of humanlysozyme is therefore preferred for use on an industrial scale. Thepotency of both natural human breast milk-derived lysozyme andrecombinant human lysozyme derived from transgenic rice was compared.

Purified natural human breast milk lysozyme (L-6394; Sigma-Aldrich,Madrid, Spain) had 101,000 units/mg protein and a 10% lysozyme content.Purified recombinant human transgenic rice lysozyme (159-53LZ-90P;Ventria Bioscience, Sacramento, Calif.) had 187,000 units/mg protein anda 90% lysozyme content. The unit definition for both lysozymes is asfollows. One unit produces an increment of the absorbance at 450 nm of0.001 per minute at pH 6.24 at 25° C., using a suspension of Micrococcuslysodeikticus as substrate, in a 2.6 ml reaction mixture (1 cm lightpath) (Shugar et al. (1952) Biochem. Biophys. 8:302-309). In order forthe results of the experiment to be comparable, the proportion 594 unitsof human lysozyme/μg GST-Tp17 of each was used. A hemoaggulutinationreaction, as detailed in Example 8, was performed, adding an equivalentamount of either the natural or recombinant lysozyme to thehemagglutination syphilis reagent. The potency of both lysozymes wastested against positive and negative syphilis samples.

As illustrated in FIGS. 17A and 17B, 6 out of the 10 samples testedshowed an improvement in sensitivity of the assay with the addition ofeither natural (LysN) or recombinant (LysR) human lysozyme.

FIG. 18A illustrates that the addition of recombinant human lysozyme toa hemagglutination syphilis reagent substantially improved the assaysensitivity. In an evaluation of 34 real positive samples, 14 of the 19samples that tested negative using the reagent that did not containlysozyme, were in fact positive when lysozyme was added to the reaction.As illustrated in FIG. 18B, the visual discrimination between a positiveand negative reaction is simplified when recombinant human lysozyme isadded to the reagent.

FIG. 19 illustrates the performance (relative hemoaglutinationintensity) of two hemagglutinantion reagents, with (reagent LysR) orwithout (reagent R) recombinant human lysozyme (LysR). In thatexperiment the LysR hemagglutination reagent out-performed the reagentR, displaying for all the sera tested (μ=34) a relative agglutinationtiter of between about 1- and about 2-fold higher.

FIG. 20 illustrates that the addition of recombinant human lysozyme(LysR) to a hemagglutination syphilis reagent does not give falsepositives results. In an evaluation of 200 blood bank samples the meanresult and standard deviation between both reagents was very close. Thepotency of recombinant human lysozyme is thus equivalent to that ofnatural breast milk lysozyme. Recombinant lysozyme can thereforesubstitute for natural lysozyme for diagnostic applications.

EXAMPLE 10 Human Lysozyme Improves the Sensitivity of Enzyme-LinkedImmunosorbent Assay (ELISA) Kits Used for Syphilis Diagnostics

Syphilis screening is routinely performed using enzyme-linkedimmunosorbent assays (ELISA) kits. These kits detect the binding ofanti-syphilis antibodies to T. pallidum antigens immobilized in thewells of a microtiter plate. Detection of the binding of anti-syphilisantibodies to the plate is normally carried out by means of either anenzyme-labeled antiserum directed against human Fc antibody chains(second generation kit) or an enzyme-labeled T. pallidum antigen (thirdgeneration kit).

Examples of such commercially available diagnostic reagents are listedbelow: Product name Manufacturer Kit Format Bioelisa Syphilis BiokitS.A. (Lliça d' Amunt, Spain) 2^(nd) generation Bioelisa Syphilis BiokitS.A. (Lliça d' Amunt, Spain) 3^(rd) generation 3.0 ICE* Syphilis AbbotMurex (Dartford, UK) 3^(rd) generation Syphilis EIA480 New MarketLaboratories 3^(rd) generation (Kentford, UK) Enzywell Diesse (Siena,Italy) 3^(rd) generation

Detection of Syphilis Antibodies Using a Second Generation Kit Format

All the reagents used in this study are provided by the commercialBioelisa Syphilis 3.0 detection kit (Biokit S.A., Lliça d'Amunt, Spain).The protocol used is that recommended in the package insert of the kit.Briefly, serum or plasma samples are added to the wells of a microtiterplate. If antibodies specific for T. pallidum are present in the sample,they form stable complexes with the antigens on the well. After washingto remove the unbound material, a rabbit conjugate anti-human IgG andanti-human IgM labelled with horseradish peroxidase is added and, if theantigen/antibody complex is present, the conjugate binds to the complex.After a second wash, an enzyme substrate solution containing a chromogenis added. This solution develops a blue color if the sample is positive.The blue color changes to yellow after blocking the reaction withsulphuric acid. The intensity of color is proportional to the anti-Tpallidum antibody concentration in the sample. Wells containing negativesamples remain colorless.

Detection Of Syphilis Antibodies Using a Third Generation Kit Format

All the reagents used in this study are provided by the commercialBioelisa Syphilis 3.0 detection kit (Biokit S.A., Lliça d'Amunt, Spain).The protocol used is that recommended in the package insert of the kit.Briefly, the test is performed by incubating test specimens in the wellsof a microtiter plate coated with T. pallidum proteins (e.g.,recombinant Tp15, Tp17 or Tp47 antigens). The specific IgG and IgMantibodies present in the sample bind to the solid-phase antigens.Subsequently, the wells are washed to remove residual test sample and T.pallidum antigens conjugated with the enzyme peroxidase are added. Theconjugate binds to the captured specific antibodies. After anotherwashing to eliminate unbound material, a solution of enzyme substrateand chromogen is added. This solution develops a blue color if thesample contains anti-T. pallidum antibodies. The blue color changes toyellow after blocking the reaction with sulphuric acid. The intensity ofcolor is proportional to the anti-T. pallidum antibody concentration inthe sample.

Addition of Recombinant Human Lysozyme

For the second generation ELISA format, recombinant human lysozyme(Ventria Bioscience, Ventura, Calif.) was added at 2.86 μg/ml to theBioelisa Syphilis 3.0 ELISA plates. For the third generation kit,recombinant human lysozyme was added either in the sample dilutionbuffer at a concentration of 10.8 μg/ml, or in the conjugate dilutionbuffer at a concentration of 1.08 μg/ml.

As represented in FIGS. 21A and 21B, when syphilis-positive human serawere tested with the Bioelisa Syphilis (second generation) and BioelisaSyphilis 3.0 (third generation) ELISA reagents, a significant gain insignal intensity was detected in the presence of human lysozyme. Theincrease in signal intensity was in the range of about 20% when lysozymewas added to the plate and sera tested in the second generation format,and in the range of about 200% (for 3 out of 4 sera) when lysozyme wasadded both in the sample dilution buffer and in the conjugate dilutionbuffer and processed according to the third generation format.Interestingly, the addition of lysozyme does not modify the response ofsyphilis-negative human sera. These data demonstrate that human lysozymeincreases the sensitivity of ELISA-based syphilis detection andscreening.

EXAMPLE 11 Purification of the Human Lysozyme/Tp17 Complex

The Human Lysozyme/Tp17 protein complex is highly antigenic, and, asdescribed above, it is useful in diagnostic assay. Isolation of purifiedHuman Lysozyme/Tp17 complex facilitates the performance of structuralstudies, such as protein crystallization and determination of the atomicstructure via X-ray diffraction. Using the experimental proceduresdescribed below, mg quantities of purified Human Lysozyme/Tp17-HIScomplex were obtained.

Isolation of Tp17-HIS Monomer and Tp17-HIS Dimers

Purified Tp17-His protein, obtained as described in example 1, wasfurther purified via gel filtration chromatography in order to separatemonomers and dimers from other aggregated forms (FIG. 22). An HR 16/50column (1.6 cm diameter×60 cm height; Amersham Biosciences, Cerdanyola,Spain) filled with 100 ml of Superose 12 HR was equilibrated with twocolumn volume of gel filtration buffer (Tris 20 mM NaCl 300 mM, pH8.0).A 2 ml sample containing 8 mg of Tp17-HIS fusion protein was theninjected and chromatography was performed at a flow rate of 1-1.25ml/min under a constant pressure of pressure 0.70 MPa Fractions (1.5 ml)corresponding to Tp17-HIS monomers and Tp17-HIS dimers were collectedfrom three consecutive chromatographic runs, and then were pooled intoan 8 ml Tp17-HIS monomeric fraction and a 6 ml Tp17-HIS dimericfraction.

Formation of the Tp17-HIS/Human Lysozyme Complex In Vitro

Recombinant human lysozyme (commercially obtained from VentriaBioscience, Sacramento, Calif., USA) consisted of highly homogenousmonomeric proteins as shown in by assay result depicted FIG. 23.

To form a complex in vitro, 3.8 mg of purified Tp17-HIS monomers andTp17-HIS dimers were incubated separately for 30 minutes at 22° C.without agitation in the presence of a 2-fold molar excess of purifiedrecombinant human lysozyme (Ventria Bioscience, Sacramento, Calif.,USA).

Purification of the Tp17-HIS/Human Lysozyme Complex by Gel Filtration

As shown in FIG. 24, the human lysozyme/Tp17 complexes were separatedfrom non-assembled monomers by gel filtration through Superose 12 HR16/50 gel as described above. The fractions corresponding to theTp17-HIS/Human lysozyme peak were collected and concentrated usingCentricon Plus 20 (UFC2LGC08, The Millipore Corporation, Bedford, Mass.,USA) devices with a 10 kDa cut-off. Buffer was exchanged duringcentrifugal filtration by washing with three column volumes (10 ml) ofTris-HCl 10 mM, NaCl 50 mM, pH 8.0. Final samples ofTp17-His_(monomers)/human lysozyme and Tp17-His_(dimers)/human lysozymewere recovered at 1.62 mg/ml and 1.57 mg/ml, respectively. Final proteinrecovery yield was 60% for Tp17-His_(monomers)/human lysozyme complexesand 44% for Tp17-His_(dimers)/human lysozyme complexes.

EXAMPLE 12 Method for Discovering Novel Tp17-Like Proteins

Tp17 and Tp17-like proteins share the capacity to bind human lysozyme.Based upon this observation, generic method were devised that providefor the detection of Tp17-like proteins in total protein extracts frompathogenic organisms. In one embodiment, human lysozyme conjugated witha detectable marker is hybridized to a protein sample. Binding of thelysozyme identifies a Tp17-like polypeptide in the sample.

Preparation of a Human Lysozyme-Horseraddish Peroxidase (HuLYS-POD)Conjugate

Human Lysozyme-Horseraddish Peroxidase conjugates were produced bymixing 9.72 mg of recombinant human lysozyme (159-53LZ-90P; VentriaBioscience, Sacramento, Calif., USA) and 13.6 mg of activated peroxidase(POD) (1.428.861; Roche, Mannheim, Germany) in a total volume of 3.5 mlcarbonate buffer (Sodium carbonate/-hydrogenocarbonate 50 mM, pH 9.55).The conjugation reaction was carried out for 2 hours at 25° C. in awater bath with manual agitation every 30 minutes. The reaction wasterminated by the successive addition of 364 μl of Triethanolamine 2M(108379; Merck, Darmstadt, Germany), and 455 μl of Sodium borohydride(45,288-2; Sigma Aldrich Chemie, Steinheim, Germany) at a concentrationof 4 mg/ml in deionized water. The resulting solution was agitatedmanually for 15 seconds and then incubated for 30 minutes at 2-8° C.Then, 227 μl of Triethanolamine 2M was added and the mixture wasincubated for 2 hours at 2-8° C. Next, 91 μl of Glycine 1 M (104201;Merck, Darmstadt, Germany) was added and the solution was dialysed for15 hours against TSG buffer (Tris-HCl 20 mM, NaCl 150 mM, Glycine 10 mM,pH 7.5). The solution was then clarified by centrifugation for 30minutes at 15.000 g. 4.5 ml of supernatant was recovered. 500 μl of BSA(Pentex Miles Inc., Kankakee, Ill., USA; 10% weight/volume in TSG buffer(Tris-HCl) and 25 μl of merthiolate-gentamycine sulfate were added tothe conjugate. Merthiolate-gentamycine sulfate was prepared bydissolving 25 mg of Gentamycine sulfate (22191 E1; Jescuder, Terrassa,Spain) and 400 mg of Thimerosal (T-5125 Sigma, Saint Louis, Mo., USA) in8.0 ml of fetal calf serum. The pH was then adjusted to pH 8.0 withsodium hydroxide (5M). The conjugate was then sterile filtered throughMillex GV a 0.22 μn filter (SLGV R04 NL; The Millipore Corporation,Bedford, Mass., USA) and was stored protected from light at 2-8° C.until use.

Processing of the Protein Sample from the Pathogen

A 15 μl sample containing 2 to 20 μg of pathogen derived protein ismixed with 5 μl of NuPAGE® LDS loading buffer (4×) (Invitrogen,Carlsbad, Calif., USA) and immediately separated on a 4-12% NuPAGE®Novex Bis-Tris polyacrylamide gel (NPO323BOX; Invitrogen, Carlsbad,Calif., USA) using NUPAGE® MES SDS running buffer (1×) (NP0002;Invitrogen, Carlsbad, Calif., USA). The applied voltage is 165 Voltsconstant for 35 minutes. After allowing the proteins to migrate, the gelis recovered and incubated for 15 minutes in electrotransfer buffer (ETbuffer: 25 mM Tris, 192 mM Glycine, 10% Methanol, no pH adjustment). Theseparated proteins on the gel are then electrophoretically transferredto an IMMOBILON® P membrane (IPVH00010; The Millipore Corporation,Bedford, Mass., USA). The membrane was prepared by rinsing once withmethanol and twice with deionized water. For performing electrophoretictransfer, the applied voltage was held constant at 65 volts for onehour.

HuLYS-POD Protein Overlay Assay

After electrotransfer, the membrane is incubated 1 hour at 22° C. inTBST (Tris-HCl 10 mM pH8.0, NaCl 150 mM, Tween 20) supplemented by 5%(weight/volume) of SVELTESSE® skimmed milk (Nestlé España, Barcelona,Spain). The membrane is next rinsed with TBST and then incubated for 1hour at 22° C. in TBST-milk containing the HuLYS-HRP conjugate, which isdiluted 1/5000. After incubation the membrane is washed three times inTBST. Immunostaining is then achieved by immersing the membrane in 10 mlof 3,3′,5,5′-Tetramethylbenzidine (TMB) (T0565; SIGMA-ALDRICH Inc.,Saint-louis, MO, USA) to visualize the proteins. As a positive control,a single band migrating at 17 kDa and corresponding to Ivy is detectedin an Escherichia coli BL21 (DE3)cell extract.

EXAMPLE 13 Gram-Positive Bacterial Pathogens Secretes Human LysozymeInhibitors

Gram positive bacteria lack an outer membrane and display to the culturemedium a thick peptidoglycan layer. As a consequence, these bacteria arelikely to be more sensitive to lysozyme killing than Gram negativebacteria. Thus, we hypothesized that Gram positive bacteria that arehuman pathogens possess lysozyme inhibitors. Due to the absence of anouter-membrane, these inhibitors may be present in the bacterial culturemedium. As described below, we assayed for the presence of lysozymeinhibitors in the culture medium from eight clinically relevantGram-positive bacterial species (Enterococcus faecalis strain CECT184,Enterococcus faecium strain ATCC10541, Staphylococcus aureus strainATCC11632, Staphylococcus epidermidis strain ATCC12228, Streptococcuspneumoniae strain ATCC49619, Streptococcus pyogenes strain CECT598,Streptococcus agalactiae strain CIP105451, and Propionibacterium acnesstrain NMR-GF).

These strains were grown for 48 hours at 37° C. in cation-adjustedMueller-Hinton broth (CAMHB). The bacterial cells were pelleted bycentrifugation (20 minutes, 14.000 g, 4° C.) and 1 ml of supernatant wasconcentrated to a final volume of 200 μl using Microcon® YM-10centrifugal filter devices (The Millipore Corporation, Bedford, Mass.,USA). Fifteen μl of each supernatant concentrate was analyzed bySDS-PAGE electrophoresis using a NuPAGE® 4-12% gradient gel and thensilver-stained (SilverXpress® kit) to visualize proteins according tothe manufacturer's instructions (The Invitrogen company, Prat deLlobregat, Barcelona, Spain).

As shown by the assay result depicted in FIG. 25, distinct proteinpatterns were detected for each of the eight bacterial species tested.The bacteriolytic activity of human lysozyme (1 unit) was determined inthe absence, or in the presence of 10 μl of the concentrated supernatantcorresponding to each of the eight bacterial species. The assay wascarried out as previously described, using the EnzCheck® Lysozymeaccording to the manufacturer's instructions (Molecular Probes, Eugene,Oreg., USA). As shown in FIG. 26, complete inhibition of lysozymeenzymatic activity was observed in the presence of Propionibacteriumacnes, Streptococcus agalactiae, and Streptococcus pyogenes concentratedsupernatants. Partial inhibition was observed in the presence ofStaphylococcus aureus, Enterococcus faecalis, and Streptococcuspneumoniae concentrated supernatents. No inhibition was detected inEnterococcus faecium and Staphylococcus epidermidis concentratedsupernatants under these assay conditions.

These data demonstrate that uncharacterized inhibitors of human lysozymeare present in clinically-relevant Gram positive bacteria, which can bea potential source for discovering such inhibitors. These inhibitorsrepresent novel and attractive targets for drugs and vaccines, as wellas for novel diagnosis assay. These experiments also indicate that Grampositive bacteria can be used to screen for wild-type or mutantbacterially expressed lysozyme polypeptides that are resistant toTp17-like polypeptide inhibition.

EXAMPLE 14 Methods of Identifying Candidate Compounds that InhibitTp17/Lysozyme Complex Formation or Stability

Tp17 and Tp17-like proteins are expressed by numerous human and animalpathogens (e.g., virus, bacteria, fingi, and protozoa). Drugs thatinterfere with the formation of and/or the stability of HuLYS/Tp17-likeprotein complexes are promising antimicrobial candidate compounds thatcan be used to prevent or treat a pathogen infection in a subject. Inorder to isolate such anti-microbial molecules, an in vitro screeningtest was devised as described below. It will be apparent to one skilledin the art that this test is useful for high throughput screening ofbiological extracts and chemical libraries. As shown in FIG. 27, anexemplary method involves the following steps: Preparation of reagents

PBS 10× was prepared by dissolving 29 g of Na₂HPO₄/12H₂O, 2 g of KH₂PO₄and 80 g of NaCl in 1 liter of distilled, apyrogenic water and adjustingthe pH to pH 6.8 with sodium hydroxide (10M). BSA-Glycine solution wasprepared by dissolving 10 g of BSA (81-003; Pentex Miles Inc., Kankakee,USA), 7.5 g of Glycine (500190; Merck, Darmstadt, Germany) and 1 g ofsodium azide (6688; Merck, Darmstadt, Germany) in 1 liter of distilledwater (MilliQ grade, The Millipore Corporation, Bedford, Mass., USA).The pH of the resulting solution was to pH 7.4 with NaOH (10M). ELISAwash buffer 10×, TMB chromogenic substrate and TMB dilution buffer arecommercially available (BioELISA ANTI-HBS 96 wells kit, 3000-1101;biokit SA, Lliça d'Amunt, Spain). ELISA sample dilution buffer wasobtained from BioELISA Syphilis 3.0 (3000-1148; biokit SA, Lliçad'Amunt, Spain).

Immobilization of Tp17 and/or Tp17-Like Protein on a Solid Substrate

Purified GST-Tp17 protein, produced as described in example 1, wasresuspended in PBS 1× at a concentration of 0.4 μg/ml and 150 μl of thatsolution was dispensed in 96 wells microtiter plates (MaxiSorp™Lockwell™; NUNC, Roskilde, Denmark) and incubated during 16 hours atroom temperature (22° C.). Next, 100 μl of BSA-Glycine solution wasadded and incubated for 1 hour at 22° C. The wells were then emptied byaspiration and filled immediately with 200 μl of BSA-sucrose. Incubationwas performed for 1 hour 30 minutes at 22° C. The liquid contents ofeach well was carefully aspired and the plates were then air-dried for 3hours in a dry room (15% relative humidity) before being packaged inair-tight plastic bags.

Contacting Tp17 and/or Tp17-Like with Chemical(s) and HuLYS-POD

100 μl of candidate compound (dissolved at a concentration ranging from10 ng/ml to 10 mg/ml in ELISA sample dilution buffer) is added to amicrowell and incubated 15 minutes at 37° C. 50 μl of HuLYS-POD diluted1/20.000 in ELISA sample dilution buffer is added to the well andincubated for an additional period of 15 minutes. The wells are thenwashed four times with wash buffer.

Color Development and Scoring of Candidate Compounds

100 μl of Horseradish peroxidase (POD) chromogenic substrate diluted insubstrate dilution buffer is then added to each well and colordevelopment is stopped after 30 minutes after addition of 100 μl ofH₂SO₄ 2M. In this experiment the average absorbance of three wellsincubated without candidate compound is normalized to 100% value.Molecules or biological extracts able to reduce the binding of HuLYS-PODbelow at least 40%, 50%, 60%, 75%, 85%, 95%, or more are considered as“Hit” candidates as evidenced by respective reductions in the signalchannel compared to the normalized value. Preferably binding is reducedbelow 85% relative to control wells not contacted with a candidatecompound.

EXAMPLE 15 Tp17 Inhibits Commercially Available Therapeutic Preparationsof Lysozyme

Chicken Lysozyme is a potent antimicrobial enzyme active against bothbacteria and viruses. Commercial preparations that contain Lysozyme asan active agent include, but are not limited to, LIZIPAINA® (BoehringerIngelheim, San Cugat del Vallés, Spain) and LISOZIMA CHIESI (CHIESIESPAÑA SA, Barcelona, Spain). Since the Tp17 protein was shown toinhibit the enzymatic antibacterial activity of purified chickenlysozyme in vitro, we postulated that Tp17 may also inhibit theantibacterial activity of LIZIPAINA® and LISOZIMA CHIESI. To test thishypothesis, one tablet of each drug, LIZIPAINA® (containing 5 mg chickenlysozyme per tablet) and LISOZIMA CHIESI (containing 250 mg chickenlysozyme per tablet), was resuspended in deionized water (The MilliporeCorporation, Bedford, Mass., USA) to adjust the Lysozyme concentrationto 4 μg/ml. These samples were then incubated in absence or in presenceof increasing amount of either GST-Tp17 or Tp17-HIS. In this experimentthe molar ratio between chicken lysozyme and its cognate inhibitor wasvaried from 1:5 to 1:50 for GST-Tp17 and from 1:16 to 1:160 forTp17-HIS. After an incubation period of 5 minutes at 20° C. the sampleswere assayed for lysozyme enzymatic activity using the EnzCheck®Lysozyme assay kit (E-22013; Molecular Probes Inc., Eugene, Oreg., USA)as recommended by the supplier guidelines.

As reported in FIGS. 28A and 28B, both GST-Tp17 and Tp17-HIS stronglyinhibited the enzymatic antibacterial activity of LIZIPAINA® andLISOZIMA CHIESI respectively. Thus, Tp17 and Tp17-like proteins arecapable of inhibiting the enzymatic antibacterial activity of twocommercially available antimicrobial drugs. It is likely that some ofthe therapeutic efficacy provided by LIZIPAINA®, LISOZIMA CHIESI, andrelated drugs is lost in vivo due to Tp17 and Tp17-like proteininhibition. As discussed above, Tp17 and Tp17-like proteins are widelyexpressed by bacterial, viral, fungal and parasite human pathogens.Consequently, lysozyme variants that are not subject to enzymaticinhibition by Tp17 and Tp17-like proteins are a promising class of novelantimicrobial drugs.

EXAMPLE 16 The Antibacterial Activity of r-Lysozyme™ is Not Inhibited byTp17

In the search of a lysozyme variant able to resist the inhibition ofTp17 and Tp17-like proteins, we tested the commercially availablerLysozyme™ (71110-5; Novagen® MerckKGaA, Darmstadt, Germany). rLysozyme™is a highly purified recombinant lysozyme that is recommended for thelysis of E. coli cells. The enzyme catalyzes the hydrolysis ofN-Aetylmuramide linkages in the bacterial cell wall. The specificactivity of rLysozyme solution (1.700.000 U/mg) is 250 times greaterthan that of chicken egg white lysozyme and therefore less enzyme isrequired to achieve E. coli Lysis. In addition, rLysozyme™ is optimallyactive at a physiological pH (6.0-8.0) that is compatible with Novagen'sline of protein and nucleic acids extraction reagents.

In that context, we compared the antibacterial activity of humanlysozyme (Ventria Bioscience, Sacramento, Calif., USA) and rLysozyme™using the EnzCheck Lysozyme assay (E-22013; Molecular Probes, Eugene,Oreg.). We first determined that under the manufacturer's prescribedassay conditions, 1 unit of human lysozyme produced the same intensityof fluorescence as 230 units of rLysozyme™. Next, we measured thefluorescence intensity of either 1 unit of human lysozyme or 230 unitsof rLysozyme™, in the presence or absence of 10 μg of either GST-Tp17 orTp17-HIS. As shown in FIG. 29, rLysozyme™ was not susceptible to theinhibition of GST-Tp17 or Tp17-HIS, while the antibacterial activity ofhuman lysozyme was strongly inhibited. Interestingly, we reproduciblyobserved a slight increase (in the range of 20%) of the rLysozyme™antibacterial activity in presence of Tp17. Taken together, theseresults suggest that rLysozyme™ is not susceptible to inhibition by Tp17and possibly Tp-17 like proteins. As a consequence, rLysozyme™represents a novel and promising antimicrobial drug that couldeventually substitute for chicken lysozyme in various therapeuticpreparations intended for human and animal use. Additionally, it can beused as an antiseptic and/or as a food preservative.

EXAMPLE 17 The Alzheimer Precursor Protein (βAPP) Shares StructuralSimilarity with Tp17-Like Proteins

Among the proteins that share extensive sequence similarity with Tp17(see, for example, FIG. 1), we identified the human Beta AmyloidPrecursor Protein (β-APP). This protein is of key medical and diagnosticinterest since it is involved in the generation of amyloid depositionduring Alzheimer disease. As shown in FIG. 30, we have discovered a wellconserved peptide motif that is shared by members of the Ivy proteinfamily, Tp17, and the three splicing isoforms of β-APP (APP770, APP751 &APP695). Manually performed multiple alignments allowed us to group allthese peptide sequences under the following consensus: (SEQ ID No: 178)CX(1,5)[KRH][AG][KRH]X(0,2)[KR]X(0,1)[EDQN]C

In this consensus, C, K, R, H, A, G, E, D, Q and N correspond to theone-letter amino acid code for Cysteine, Lysine, Arginine, Histidine,Alanine, Glycine, Glutamic acid, Aspartic acid, Glutamine, andAsparagine, respectively. The syntax rules are the one used in thePROSITE database. This consensus can also be represented as follows: CysXaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ (SEQ ID NO: 177) Xaa₆ Xaa₇ Xaa₈ Xaa₉ Xaa₁₀Xaa₁₁ Xaa₁₂ Xaa₁₃ Cyswhere Xaa₁, Xaa₂ Xaa₃ Xaa₄ and Xaa₅ are any amino acid or are absent,Xaa₆ is amino acid K, R or H, Xaa₇ is A or G, Xaa₈ is K, R, or H, Xaa₉and Xaa₁₀ are any amino acid or are absent, Xaa₁₁ is amino acid K or R,Xaa₁₂ is any amino acid or is absent, and Xaa₁₃ is amino acid E, D, Q orN.

For three of these proteins (Ivy from E. coli, Tp17, and P. aeruginosa),crystal structures were available. Using the Cn3D molecular viewer, wefound that the related peptide stretches of each of the three proteinsexhibited a conserved three dimensional organization. As shown in FIG.31, all three form an extended beta sheet, displaying a disulfidebridged peptide loop. These proteins share a conserved three dimensionalstructural organization. All three molecules contain an extended betasheet structure and an exposed disulfide-bridged peptide loop. Thisstructural motif is conserved in all three isoforms (APP770, APP751 &APP695) of human β-APP protein. The 3D views were generated using Cn3Dsoftware, which is available on the National Center for Biotechnology'swebsite. and the following Protein Data Bank (PDB) atomic coordinatesfiles: 1GPQ (E. coli Ivy), 1UUZ (P. aeruginosa Ivy) and 1MWP (Homosapiens β-APP). These data suggest that Ivy, Tp17, and β-APP arestructurally and functionally related.

EXAMPLE 18 Human Lysozyme Binds to Immobilized Alzheimer PrecursorProtein (βAPP)

As shown in FIG. 32, the lysozyme binding site identified in β-APP liesat the extreme N-terminus of the protein and spans residues 92 to 116.The isoform APP751 (751 amino acid long) is an exemplary APP-likepolypeptide. The conclusions drawn from this figure are also true forisoforms APP695 and APP770. The amino acid numbers refer to amino acidpositions in the β-APP sequence deposited under Genbank accession numberNP_(—)958816 (SEQ ID NO:313). The lysozyme binding site is locatedbetween residues 92 to 116. Specifically, we used a longer fragment ofβ-APP derivative, called sAPPα and a shorter fragment called sAPPα(304-612): Both sAPPα and sAPPα (304-612) are derived from the APP695isoform and consequently lack the kunitz protease inhibitor domain(residues 289 to 342 in APP770 and APP751).

To test whether the β-APP protein binds huLYS, we performed lysozymebinding assays using purified, recombinant forms of β-APP. Both sAPPα(product number: S9564) and sAPPα (302-612) (product number: S8065) werepurchased from Sigma-Aldrich (Tres cantos, Madrid, Spain). 10 μg of eachprotein was spotted onto a Nytran membrane (Scleicher & Schüell, Dassel,Germany). Non-specific adsorption sites were blocked by incubation forone hour in TBST-milk. After three washes with TBST, the membrane wasincubated for one hour with horse radish peroxidase (POD)-labeled humanlysozyme diluted 1/1000 in TBST-milk. Next, the membrane was washedthree times, for 5 minutes each wash, with TBST. Staining was performedby adding 5 ml of POD chromogenic substrate (TMB, product number: T0565,Sigma). As shown in FIG. 33, huLYS bound to sAPPα, but not to sAPPα(302-612). This observation suggested that the 1-APP protein binds tohuLYS, and that the binding site is located in the N-terminal region,between residues 92 and 116 of 1-APP. These data define a novelpharmacological target for the treatment of Alzheimer disease. Moreover,the 1-APP/huLYS protein complex likely serves as a diagnostic marker ofAlzheimer's disease.

Diagnostic compositions and methods related to Alzheimer's diseasefeature fragments of a substantially pure APP-like polypeptidecomprising an amino acid sequence of Cys Xaa₁ Xaa₂ Xaa₃ Xaa4 Xaa₅ Xaa6Xaa₇ Xaa₈ Xaa₉ Xaa₁₀ Xaa₁₁ Xaa₁₂ Xaa₁₃ Cys (SEQ ID NO:177), where Xaa₁Xaa₂ Xaa₃, Xaa4 and Xaa₅ are any amino acid or are absent, Xaa6 is aminoacid K, R or H, Xaa₇ is A or G, Xaa₈ is K, R, or H, Xaa₉ and Xaa₁₀ areany amino acid or are absent, Xaa₁₁ is amino acid K or R, Xaa₁₂ is anyamino acid or is absent, and Xaa₁₃ is amino acid E, D, Q or N. Thecompositions and methods also feature as nucleic acid molecules encodingthis polypeptide fragments, vectors for the expression of suchfragments, and host cells containing these vectors. Such fragments maybe expressed as recombinant polypeptides and used for the generation ofantibodies that recognize the fragment or that recognize the fragmentwhen complexed to a lysozyme polypeptide.

Antibodies that specifically bind to an APP-like polypeptide/lysozymecomplex are useful in methods for diagnosing Alzheimer's disease in asubject, where the antibody is used to probe a biological sample fromthe subject, such as a serum sample, a cerebrospinal fluid sample, or atissue sample. At present, a definitive diagnosis of Alzheimer's diseasegenerally requires a post-mortem examination of brain tissue from asubject. Thus, the present compositions and methods, which facilitatethe diagnosis of Alzheimer's disease in bodily fluids obtained from aliving subject, provide a significant improvement over existingdiagnostic methods.

Specifically, the present invention provides a method of diagnosingAlzheimer's disease by detecting the presence in a sample from thesubject of a complex between an APP-like polypeptide and a lysozymepolypeptide, or the presence of an antibody that binds to such acomplex. Further, a method is provided for identifying a candidatecompound that modulates binding between the APP-like polypeptide and alysozyme polypeptide through detecting a reduction in binding betweenthe Tp17-like polypeptide and the lysozyme polypeptide in the presenceof the candidate compound.

EXAMPLE 19 Computer Identification of Novel Tp17-Like Proteins

Peptide sequence databases (Swissprot & TrEMBL) were searched forproteins that share the CX(1,5)[KRH][AG][KRH]X(0,2)[KR]X(0,1)[EDQN]C(SEQ ID NO:178) consensus sequence defined in FIG. 30. The bacterial,viral and eukaryotic proteins identified as having this sequence areshown in FIGS. 34A and 34B. These proteins represent novel therapeutictargets and immunogenic compositions that will likely interfere with theformation of Tp17-like/huLYS protein complexes. Moreover, theseproteins, complexed with huLYS, may represent attractive markers for thediagnosis of human, animal and plant diseases.

EXAMPLE 20 Inhibition and Binding Capacity of Tp17 Mutants

To determine whether H31 and His 104 of Tp17 contributed to huLYSinhibition, we performed site-directed mutagenesis of the gene encodingGST-Tp17 (FIG. 35A). The H₃₁A and His₁₀₄A GST-Tp17 mutant polypeptideswere generated by introducing two nucleotide changes in the pGEX2T-Tp17plasmid vector using the QuickChange® Site-directed mutagenesis kit(Stratagene, La Jolla, Calif., USA). For that purpose, mutagenicoligonucleotides Fw_CPAAG (5′-CCG TGT GTC CGG CCG CCG GGA AGG C-3′)(SEQ. ID. NO:298) and Bw_CPAAG (5′-GCC TTC CCG GCG GCC GGA CAC ACG G-3′)(SEQ. ID. NO: 299)and oligonucleotides Fw_KPAE (5′-AAT CGA AGG CAC CGGCCG AGA AAG AGC TGT ACG-3) (SEQ. ID NO: 300) and Bw_KPAE (5′-CGT ACA GCTCTT TCT CGG G GTG CCT TCG ATT-3′) (SEQ. ID NO: 301) were separatelyannealed to denatured pGEX2T-Tp17, and extended using Pfu Turbo DNApolymerase under conditions recommended by the supplier. Non-methylatedDNA template was eliminated by DpnI digestion and the resultingdigestion mixture was used to trasform XL-1 Blue® supercompetent cells.Mutant plasmids were selected by restriction mapping the resulting PCRamplicons. The presence of the H₃₁A mutation was confirmed by sequencingthe full-length mutated Tp17 gene. Mutant GST-Tp17 carrying the H₃₁A orthe His₁₀₄A mutation was then produced and purified as described inExample 1. Lysozyme inhibition assays were performed as described below.

Each 100 μl reaction mixture contained 25 μl of lysozyme solution (2units for human lysozyme, dissolved in reaction buffer), 25 μl of a testprotein solution, and 50 μl of a suspension of fluorescein-labeledMicrococcus lysodeikticus (50 μg/ml). The reaction mixture was thenincubated for 45 minutes at 37° C. Fluorescence present in the reactionmixture was measured (λ_(exc.)=485 mm; λ_(emi.)=520 nm) using afluorescence multi-well plate reader model FLx800 (Bio TEK instruments,Winooski, Vt.) equipped with KC junior data acquisition software (BioTEK Instruments, Winooski, Vt.). The resulting fluorescent data, shownin FIG. 35B, represents the mean and standard deviation corresponding tothe reading of three independent wells. A negative control wellcontained 50 μl of deoinized water and 50 μl of Fluorescein-labeledMicrococcus lysodeikticus suspension.

As shown in FIGS. 35A and 35B, H₃₁A and His₁₀₄A mutant polypeptidesshowed a decrease in lysozyme inhibition, but retained their ability tobind lysozyme (FIG. 36). These data demonstrated that amino acidpositions 31 and 104 contribute to lysozyme binding and/or inhibition.Therefore, embodiments of a mutant Tp17 according to the presentinvention includes one or more mutations at and/or around H₃₁ and His₁₀₄of Tp-17 and corresponding sites in other Tp-17 like pathogens. Such amutant can interfere with normal inhibition of a Tp-17 like protein invivo and therefore having therapeutic effects described above.

EXAMPLE 21 Tp17 May Bind to SLLP1 Lysozyme-Like Protein

A unique, non-bacteriolytic, chicken or conventional-type (C-type)lysozyme-like protein, SLLP1, is present in the acrosome of human sperm(Mandal et al., Biol Reprod. 68:1525-37, 2003). Normally, C-typelysozymes are bacteriolytic and can bind to N-acetylglucosamines linkedby beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of20), including all the cysteines, are conserved in SLLP1, but the twocatalytic residues E35 and D52 conserved in C-lysozymes have beenreplaced in SLLP1 with T and N, respectively, to become T122 and N139.Mandal et al. hypothesized that, after acrosome reaction, SLLP1 could bea potential receptor for the egg oligosaccharide residueN-acetylglucosamine, which is present in the extracellular matrix overthe egg plasma membrane, within the perivitelline space, pores of zonapellucida, and cumulus layers.

We have found that huLYS, chkLYS and SLLP1 share more than 55% sequenceidentity. As shown in FIG. 37, while human lysozyme and chicken lysozymeshare 63% amino acid sequence identity, SLLP1 and human lysozyme share58% sequence identity. Since Tp17 is able to bind to both huLYS andchkLYS, and based on the high level of sequence identity present betweenthese proteins, it is likely that Tp17-like polypeptides including Tp17are also capable of binding to SLLP1. This interaction may facilitatepathogen transmission during sexual contact. As shown in FIG. 38, anSLLP1/Tp17-like polypeptide complex represents a promising therapeutictarget for the control of human and animal fertility. For example, asillustrated in FIG. 38, adminstering an effective amount of a Tp-17 likepolypeptide may serve as a method of contraception as it interferes withnormal docking of a spermatozoa to a cognate receptor at an egg cellsurface.

Further, candidate compounds that decrease the binding of a Tp17-likepolypeptide to SLLP1 are useful inhibitors of sexually transmitteddisease. Methods for screening for such compounds are described above.

In addition, while wild-type SLLP1 is not likely to be bacteriolytic,mutant SLLP1, containing amino acid alterations of T122 to E and/or ofN139 to D, especially the double mutant containing both mutations backto the conserved sequence of C-type lysozyme, are likely to acquirebacteriolytic activity. Mutant SLLP1 containing these mutations wouldlikely fail to bind Tp17-like polypeptides and would likely haveincreased lysozyme enzymatic activity relative to chicken lysozyme, forexample. As such, mutant SLLP1 may be used for its enhancedantimicrobial, e.g., bacteriolytic, capability as a pharmaceuticalcomposition or disinfectant. Other lysozyme variant such asbacteriophage T4's lysozyme, can also be used in a similar way. Oneembodiment of such a composition may include between about 2 to 100 mgof the lysozyme variant (preferably about 5 mg), about 2 mg of papainand/or about 3 mg of bacitracin. A preferred embodiment is dissolvablefor oral intake.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. Any aspects andfeatures of the above desribed invention can be combined. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are embraced therein.

Incorporation by Reference

All sequence access numbers, publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if the contents of each individualpublication or patent document was incorporated herein.

1. A fragment of a Tp17-like polypeptide, comprising: a lysozyme bindingmotif comprising at least four amino acids, two of the four amino acidsselected from the group consisting of Cys, Pro, His, and Arg, whereinthe lysozyme binding motif is not CKPHDC (SEQ ID NO. 24).
 2. Thefragment of claim 1, wherein the lysozyme binding motif comprises anamino acid sequence of Cys Xaa1 Xaa2 Arg Xaa3 Xaa4 Xaa5 Cys (SEQ ID NO.314), wherein Xaa1, Xaa2, Xaa3, Xaa4, or Xaa5 is any amino acid, isabsent, or is a peptide bond.
 3. The fragment of claim 1, wherein thelysozyme binding motif comprises an amino acid sequence of Cys Xaa1 Xaa2Arg Xaa3 Xaa4 Xaa5 Cys (SEQ ID NO. 314), wherein Xaa1 is Pro, Ala, Val,or Ser; Xaa2 is Gln, Glu, or His; Xaa3 is Leu or Met; Xaa4 is Ser, Ala,or Gly; and Xaa5 is Ser, Val, Ala, Lys, or Cys.
 4. The fragment of claim1, wherein the lysozyme binding motif comprises an amino acid sequenceof Cys Pro Xaa1 Arg Xaa2 Xaa3 Xaa4 Cys (SEQ ID NO. 315), wherein Xaa1 isGln, Glu, or His; Xaa2 is Leu or Met; Xaa3 is Ser or Ala; Xaa4 is Ser,Ala, or Val.
 5. The fragment of claim 4, wherein the lysozyme bindingmotif comprises an amino acid sequence selected from the groupconsisting of CPHRLSVC (SEQ ID NO. 316), CPHRLSSC (SEQ ID NO. 317),CPERLASC (SEQ ID NO. 318), CPERMASC (SEQ ID NO. 319), CPERLSSC (SEQ IDNO. 320), and CPQRLSSC (SEQ ID NO. 321).
 6. The fragment of claim 5,wherein the lysozyme binding motif comprises amino acid sequenceCPHRLSVC (SEQ ID NO. 316).
 7. The fragment of claim 1, wherein thepolypeptide is derived from the E2 envelope glycoprotein of a HepatitisC virus.
 8. The fragment of claim 1, wherein the lysozyme binding motifis affixed to a solid support.
 9. The fragment of claim 1, wherein thepolypeptide is linked to a detectable label.
 10. A substantially purenucleic acid molecule encoding the fragment of claim
 2. 11. A vectorcomprising the nucleic acid molecule of claim
 10. 12. A host cellcomprising the vector of claim
 11. 13. A fusion protein comprising thefragment of claim
 1. 14. A composition comprising a first polypeptidecomprising at least a lysozyme binding motif and a second polypeptide,wherein the lysozyme binding motif comprises at least four amino acids,two of which are selected from the group consisting of Cys, Pro, His,and Arg, wherein the first polypeptide is not the Ivy polypeptide andthe second polypeptide comprisies a lysozyme.
 15. The composition ofclaim 14, wherein the lysozyme binding motif comprises an amino acidsequence of Cys Xaa1 Xaa2 Arg Xaa3 Xaa4 Xaa5 Cys (SEQ ID NO. 314),wherein Xaa1, Xaa2, Xaa3, Xaa4, or Xaa5 is any amino acid, is absent, oris a peptide bond.
 16. The composition of claim 14, wherein the lysozymebinding motif comprises an amino acid sequence of Cys Xaa1 Xaa2 Arg Xaa3Xaa4 Xaa5 Cys (SEQ ID NO. 314), wherein Xaa1 is Pro, Ala, Val, or Ser;Xaa2 is Gln, Glu, or His; Xaa3 is Leu or Met; Xaa4 is Ser, Ala, or Gly;and Xaa5 is Ser, Val, Ala, Lys, or Cys.
 17. The composition of claim 14,wherein the lysozyme binding motif comprises an amino acid sequence ofCys Pro Xaa1 Arg Xaa2 Xaa3 Xaa4 Cys (SEQ ID NO. 315), wherein Xaa1 isGln, Glu, or His; Xaa2 is Leu or Met; Xaa3 is Ser or Ala; Xaa4 is Ser,Ala, or Val.
 18. The composition of claim 14, wherein the lysozymebinding motif comprises an amino acid sequence selected from the groupconsisting of CPHRLSVC (SEQ ID NO. 316), CPHRLSSC (SEQ ID NO. 317),CPERLASC (SEQ ID NO. 318), CPERMASC (SEQ ID NO. 319), CPERLSSC (SEQ IDNO. 320), and CPQRLSSC (SEQ ID NO. 321).
 19. The composition of claim14, wherein the lysozyme binding motif comprises an amino acid sequenceof CPHRLSVC.
 20. The composition of claim 14, wherein the firstpolypeptide is derived from the E2 envelope glycoprotein of a HepatitisC virus.
 21. The composition of claim 14, wherein the second polypeptidecomprises the following amino acid sequence: Xaa Xaa Xaa Xaa Xaa Xaa CysXaa (SEQ ID NO: 28) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa XaaXaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly XaaXaa Gln Ile Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa TrpXaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa,

wherein Xaa is any amino acid or is absent.
 22. A method for detectingan immune response against a pathogen in a subject, the methodcomprising the steps of: (a) contacting a biological sample from thesubject with an exogenous lysozyme and a polypeptide comprising alysozyme binding motif; and (b) detecting antibody binding to thepolypeptide or to a polypeptide-lysozyme complex.
 23. The method ofclaim 22, wherein the diagnostic assay is an agglutination assay. 24.The method of claim 22, wherein the lysozyme is contacted with thepolypeptide comprising the lysozyme binding motif prior to, during, orafter contacting the biological sample.
 25. The method of claim 22,wherein the lysozyme is derived from human.
 26. The method of claim 22,wherein the pathogen is Hepatitis C virus.
 27. A kit for detecting animmune response to a pathogen comprising a first polypeptide and asecond polypeptide, wherein the first polypeptide comprises at least alysozyme binding motif and the second polypeptide is a lysozyme.
 28. Thekit of claim 27, wherein the lysozyme binding motif comprises an aminoacid sequence of Cys Xaa1 Xaa2 Arg Xaa3 Xaa4 Xaa5 Cys (SEQ ID NO. 314),wherein Xaa1, Xaa2, Xaa3, Xaa4, or Xaa5 is any amino acid, is absent, oris a peptide bond.
 29. The kit of claim 27, wherein the lysozyme bindingmotif comprises an amino acid sequence of Cys Xaa1 Xaa2 Arg Xaa3 Xaa4Xaa5 Cys (SEQ ID NO. 314), wherein Xaa1 is Pro, Ala, Val, or Ser; Xaa2is Gln, Glu, or His; Xaa3 is Leu or Met; Xaa4 is Ser, Ala, or Gly; andXaa5 is Ser, Val, Ala, Lys, or Cys.
 30. The kit of claim 27, wherein thelysozyme binding motif comprises an amino acid sequence of Cys Pro Xaa1Arg Xaa2 Xaa3 Xaa4 Cys (SEQ ID NO. 315), wherein Xaa1 is Gln, Glu, orHis; Xaa2 is Leu or Met; Xaa3 is Ser or Ala; Xaa4 is Ser, Ala, or Val.31. The kit of claim 27, wherein the lysozyme binding motif comprises anamino acid sequence selected from the group consisting of CPHRLSVC (SEQID NO. 316), CPHRLSSC (SEQ ID NO. 317), CPERLASC (SEQ ID NO. 318),CPERMASC (SEQ ID NO. 319), CPERLSSC (SEQ ID NO. 320), and CPQRLSSC (SEQID NO. 321).
 32. The composition of claim 27, wherein the lysozymebinding motif comprises an amino acid sequence of CPHRLSVC (SEQ ID NO.316).
 33. The kit of claim 27, wherein the second polypeptide comprisesthe following amino acid sequence: Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa (SEQID NO: 28) Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Trp Xaa Cys Xaa Xaa Xaa Xaa Glu Ser Xaa Xaa Xaa ThrXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Asp Tyr Gly Xaa Xaa GlnIle Asn Xaa Xaa Xaa Trp Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys Xaa Xaa Xaa Cys Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys AlaLys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Xaa Xaa Trp Xaa XaaXaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa,

wherein Xaa is any amino acid or is absent.
 34. The kit of claim 27,wherein the first or the second polypeptide is attached to a solidsupport.
 35. The kit of claim 34, wherein the solid support is selectedfrom the group consisting of a resin, a gel, a bead, a well, a column, achip, a membrane, a matrix, a plate, and a filter device.
 36. The kit ofclaim 27, wherein the first or the second polypeptide is linked to adetectable label.
 37. The kit of claim 27, wherein the pathogen isHepatitis C virus. 38-42. (canceled)